Breeding evaluation method of exogenous hydrocarbonophilic emulsifying functional bacteria

Abstract

The invention belongs to the technical field of tertiary oil recovery, and particularly relates to a breeding evaluation method of exogenous hydrocarbonophilic emulsifying functional bacteria. The breeding evaluation method comprises the following steps: primarily culturing and screening exogenous hydrocarbonophilic emulsifying bacteria; evaluating the growth and emulsifying ability of the hydrocarbonophilic emulsifying bacteria in the target oil reservoir environment; hydrocarbon-philic emulsifying bacteria compatible with oil reservoir endogenous bacteria of the target oil reservoir are screened in combination with the influence of biological factors; the activated growth of the activating agent system on the hydrocarbonophilic emulsifying bacteria is studied; evaluating the utilization conversion rate of the exogenous hydrocarbonophilic emulsifying bacteria on the activator system; simulating a target oil reservoir environment, and evaluating the oil displacement effect after the exogenous hydrocarbonophilic emulsifying bacteria and an activating agent system are introduced into the rock core; constructing a screening and oil displacement evaluation method of the exogenous hydrocarbonophilic emulsifying bacteria; the invention relates to field application of exogenous hydrocarbonophilic emulsifying functional bacteria. The cost is saved, and the oil reservoir range is widened.

Description

Technical Field

[0001] The present invention belongs to the technical field of tertiary oil recovery, and particularly relates to a method for screening and evaluating exogenous hydrocarbon-utilizing emulsifying functional bacteria. Background Art

[0002] Microbial enhanced oil recovery technology utilizes the reproduction and metabolism of microorganisms in oil reservoirs to act on crude oil, thereby improving the oil recovery rate. Oil recovery functional microorganisms are the material basis for implementing microbial enhanced oil recovery technology and are also important research objects. Hydrocarbon-utilizing emulsifying functional bacteria are an important class of oil recovery functional bacteria existing in oil reservoir environments and play a starting role in crude oil metabolism. After activation, these functional bacteria trigger the exertion of oil recovery functions in the entire microbial ecosystem and promote the progress of microbial enhanced oil recovery metabolism. Previous studies have shown that in some oil reservoirs, due to the limitations of current microbial activation methods, it is difficult to directionally activate endogenous hydrocarbon-utilizing emulsifying functional bacteria from the complex endogenous microorganisms, resulting in unstable application effects of microbial enhanced oil recovery technology; in some other oil reservoirs, there is a lack of endogenous hydrocarbon-utilizing emulsifying functional bacteria, resulting in an incomplete microbial population structure after activation and restricting the application of microbial enhanced oil recovery technology. Therefore, it is considered to introduce exogenous hydrocarbon-utilizing emulsifying functional bacteria into oil reservoirs to reshape and regulate the establishment of a microbial ecosystem, increase the quantity and activity of hydrocarbon-utilizing emulsifying bacteria, and achieve the purpose of enhancing microbial enhanced oil recovery with hydrocarbon-utilizing emulsifying bacteria.

[0003] The screening and breeding of highly efficient exogenous hydrocarbon-utilizing emulsifying functional bacteria have become the key to enhancing microbial enhanced oil recovery technology. On the one hand, it is necessary to screen superior hydrocarbon-utilizing emulsifying functional bacteria, and on the other hand, a suitable activator system is required to efficiently cultivate hydrocarbon-utilizing emulsifying functional bacteria to exert their oil displacement function. However, exogenous hydrocarbon-utilizing emulsifying functional bacteria are affected by biological factors (endogenous microorganisms) and abiotic factors (crude oil, temperature, salinity, etc.) in the oil reservoir environment, and have poor adaptability to the oil reservoir environment, making it difficult to effectively colonize in the oil reservoir environment to exert their oil displacement function. Currently, the screening and activation evaluation methods for hydrocarbon-utilizing emulsifying bacteria have poor oil reservoir specificity, especially lacking indicators for characterizing the relationship between the growth of hydrocarbon-utilizing emulsifying bacteria and their ability to emulsify crude oil, evaluation indicators for the compatibility between hydrocarbon-utilizing emulsifying bacteria and endogenous bacteria in the oil reservoir environment after being introduced into the oil reservoir environment, and evaluation indicators for the conversion efficiency of hydrocarbon-utilizing emulsifying bacteria to degrade and utilize the activator system into biological emulsifiers, etc. As a result, the adaptability of hydrocarbon-utilizing emulsifying bacteria in the actual oil reservoir environment is not clear, thus unable to effectively guide the field application of exogenous hydrocarbon-utilizing emulsifying bacteria. Therefore, constructing a systematic and comprehensive screening and evaluation method for exogenous hydrocarbon-utilizing emulsifying bacteria is beneficial to improving the application effect of enhancing microbial enhanced oil recovery technology with hydrocarbon-utilizing emulsifying bacteria.

[0004] After literature retrieval, the invention patent "A Method for Improving the Reservoir Adaptability of Exogenous Functional Microorganisms" CN107795308 B discloses a method for improving the reservoir adaptability of exogenous functional microorganisms. First, exogenous functional microorganisms are screened according to the reservoir temperature and salinity; then, the exogenous functional microorganisms are domesticated in terms of temperature, salinity, anaerobic conditions, and pressure; finally, physical model experiments are carried out to screen the exogenous functional microorganisms for field tests. However, this patent has the following deficiencies: (1) After the exogenous functional microorganisms are injected into the reservoir, they will inevitably compete with the endogenous microorganisms. The evaluation of the growth and function of exogenous functional microorganisms affected by endogenous microorganisms is ignored during the screening process, and only the influence of abiotic factors in the reservoir environment on exogenous functional microorganisms is considered; (2) The physical model oil displacement experiment reflects the oil displacement effect of pure exogenous functional microorganisms, lacking the evaluation of the oil displacement effect of the entire reservoir ecosystem after the introduction of exogenous bacteria under the condition of the existence of endogenous microorganisms in the reservoir; (3) Exogenous functional microorganisms adapted to the reservoir temperature and salinity are generally screened, and no targeted breeding evaluation is carried out on exogenous microorganisms with specific oil displacement functions.

[0005] The invention patent "A Regulation Method for the In-situ Origin of Emulsifying Functional Bacteria in Reservoirs" CN105221126 B discloses a regulation method for the in-situ origin of emulsifying functional bacteria in reservoirs. First, a target reservoir lacking emulsifying functional bacteria is screened; then, emulsifying functional bacteria are screened from two aspects: the emulsifying ability of crude oil and the oil displacement effect of the physical model; finally, field implementation is carried out on the basis of determining the regulation plan for emulsifying functional bacteria. However, this patent has the following deficiencies: The screening evaluation index of the emulsified particle size of crude oil is single, and the correlation characteristics between the growth of emulsifying functional bacteria and the hydrocarbon-degrading emulsifying ability under the reservoir environment with different crude oil viscosities are not clear, making it difficult to provide targeted guidance for complex field applications.

[0006] In the article "Study on the Hydrocarbon Degradation Characteristics of Geobacillus thermophilus DM-2" by Liu Qingkun, Wang Jun, Li Guoqiang, etc., a strain of Geobacillus thermophilus DM-2 was screened from the Dagang Oilfield. This bacterium degrades crude oil hydrocarbons under the conditions of 45-70 °C, pH 4.0-10.0, and a salt ion concentration of 0.2%-3.0%, and has application potential in the biological treatment of high-temperature petroleum sewage and the exploitation of heavy crude oil. However, this article has the following deficiencies: (1) No activator system for effectively activating Geobacillus thermophilus is provided, making it difficult to ensure the effective growth and reproduction of this bacterium in the field; (2) Lack of evaluation of the compatibility and oil displacement effect between exogenous hydrocarbon-emulsifying bacteria and endogenous bacteria in the reservoir environment after the introduction of exogenous hydrocarbon-emulsifying bacteria into the reservoir environment, unable to master the dynamic changes of exogenous bacteria after being introduced into the reservoir environment, and difficult to regulate them to become dominant bacteria and play a role in the reservoir environment.

[0007] Therefore, establishing an efficient and multi-factor breeding method for hydrocarbon-emulsifying bacteria to greatly improve the matching degree of this bacterium with the actual application reservoir is not only beneficial to enhancing the effect of microbial enhanced oil recovery, but also of great significance for broadening the application scope of microbial enhanced oil recovery technology. Summary of the Invention

[0008] The object of the present invention is to overcome the defects existing in the above-mentioned prior art and provide a method for screening and evaluating exogenous hydrocarbon-emulsifying functional bacteria. The present invention mainly focuses on two aspects: screening of hydrocarbon-emulsifying bacteria and cultivation of activator systems. Among them, the hydrocarbon-emulsifying bacteria are not only required to have excellent crude oil emulsification performance, but also can be well compatible with endogenous microorganisms in the oil reservoir and colonize in the oil reservoir environment to become the dominant flora. In addition, a suitable activator system can efficiently cultivate the screened hydrocarbon-emulsifying bacteria, convert effective nutrient substrates into biological emulsifiers at the same time, and play a synergistic oil displacement role with endogenous bacteria after being introduced into the oil reservoir environment, thereby further improving the crude oil recovery rate.

[0009] The present invention discloses a method for screening and evaluating exogenous hydrocarbon-emulsifying functional bacteria, which is characterized in that the screening and evaluating method specifically includes the following steps:

[0010] (1) Preliminary culture and screening of exogenous hydrocarbon-emulsifying bacteria;

[0011] (2) Evaluate the growth and emulsification ability of hydrocarbon-emulsifying bacteria in the target oil reservoir environment;

[0012] (3) Combine the influence of biological factors to screen hydrocarbon-emulsifying bacteria compatible with endogenous bacteria in the target oil reservoir;

[0013] (4) Study the activation growth of activator systems on hydrocarbon-emulsifying bacteria;

[0014] (5) Evaluate the utilization conversion rate of exogenous hydrocarbon-emulsifying bacteria to activator systems;

[0015] (6) Simulate the target oil reservoir environment and evaluate the oil displacement effect after introducing exogenous hydrocarbon-emulsifying bacteria and activator systems into the core;

[0016] (7) According to the evaluation results of steps (1), (2), (3), (4), (5) and (6), construct a screening and oil displacement evaluation method for exogenous hydrocarbon-emulsifying bacteria;

[0017] (8) Field application of exogenous hydrocarbon-emulsifying functional bacteria.

[0018] Preferably, the preliminary culture and screening of the exogenous hydrocarbonophilic emulsifying bacteria in step (1) are carried out as follows: 1-2 L of the injected water and produced fluid of the target reservoir are respectively taken and injected into a sampling bucket, centrifuged and concentrated to prepare a bacterial suspension, sampled and coated on an LB medium, and strain isolation and purification are carried out at the reservoir temperature. Genomic DNA is extracted from the cultured strains, and bacterial 16S rDNA sequencing method is used for molecular biological identification of the strains to determine the types of common strains in the injected water and produced fluid. The common strains can represent the strains with strong reservoir adaptation potential that survive in the reservoir environment introduced into the injected water; then the common strains are respectively inoculated into a fermentation nutrient system and fermented for 2-3 d, and the emulsification index of the fermentation broth is measured.

[0019] Preferably, the fermentation nutrient system is 0.2-0.3 wt% sucrose, 0.2-0.3 wt% peptone, K 2 HPO 4 0.1-0.2 wt% and 0.05-0.1 wt% yeast powder.

[0020] Preferably, the evaluation of the growth and emulsification ability of hydrocarbonophilic emulsifying bacteria in the target reservoir environment in step (2) is carried out as follows: First, the screened hydrocarbonophilic emulsifying bacteria are inoculated into a crude oil plate prepared with the crude oil of the target reservoir and cultured at the target reservoir temperature for 3-5 d. The ratio (D / d) of the diameter (D) of the crude oil spreading circle of the hydrocarbonophilic emulsifying bacteria to the colony diameter (d) can represent the growth and emulsification ability of the hydrocarbonophilic emulsifying bacteria. Hydrocarbonophilic emulsifying bacteria that can grow with crude oil as the sole carbon source and effectively emulsify and strip crude oil are screened according to the D / d ratio.

[0021] Preferably, the crude oil plate is 2.0-0.3 wt% urea, NH 4 NO 3 1.0-2.0 wt%, KH 2 PO 4 0.3-0.5 wt%, K 2 HPO 4 0.3-0.5 wt%, 0.1-0.2 wt% yeast powder, 15-20 wt% agar powder, sterilized at 121 °C for 20 min, taken out and poured into plates. After the plates solidify, 10 mL of sterilized crude oil is added and spread evenly with a spreading rod.

[0022] Preferably, in step (3), the hydrocarbon-utilizing emulsifying bacteria compatible with the indigenous bacteria in the target reservoir are screened by considering the influence of biological factors. The specific steps are as follows: Add 200 mL of a 1:1 mixture of the target reservoir injection water and formation water and a fermentation nutrient system into a 500 mL culture flask, and then add 10 - 20 mL of the hydrocarbon-utilizing emulsifying bacteria culture solution. Place it in a static state at the temperature of the target reservoir for 20 - 30 days. Determine the compatibility between the exogenous hydrocarbon-utilizing emulsifying bacteria and the indigenous bacteria in the reservoir based on the population proportion of the exogenous hydrocarbon-utilizing emulsifying bacteria and the copy number of the emulsification gene in the activated reservoir ecosystem.

[0023] Preferably, in step (4), the activation and growth of the hydrocarbon-utilizing emulsifying bacteria by the activator system are studied. The specific steps are as follows: Add 1000 mL of a 1:1 mixture of the target reservoir injection water and formation water and the activator system into a 2000 mL culture flask, sterilize it, and then add 5 - 10 mL of the hydrocarbon-utilizing emulsifying bacteria fermentation broth. Place it in a culture at the temperature of the target reservoir, and conduct research on the dry cell weight and the growth stationary phase evaluation respectively.

[0024] Preferably, the dry cell weight evaluation refers to placing the culture flask in a static state at the temperature of the target reservoir for 3 - 5 days, sampling, centrifuging, discarding the supernatant, drying the cells, and weighing the cells for detection.

[0025] Preferably, the growth stationary phase evaluation refers to placing the culture flask in a static state at the temperature of the target reservoir for 60 days, detecting the OD 600nm value every 2 days, and counting the duration of the peak OD 600nm value of the hydrocarbon-utilizing emulsifying bacteria.

[0026] Preferably, the activator system refers to 0.2 - 0.5 wt% of a carbon source, 0.1 - 0.3 wt% of a nitrogen source, 0.05 - 0.1 wt% of a phosphorus source, and 0.1 - 0.2 wt% of a growth factor.

[0027] More preferably, the carbon source is one of starch, tapioca flour, dextrin, and molasses; the nitrogen source is one of corn steep liquor, soybean cake powder, peptone, urea, ammonium chloride, and sodium nitrate; the phosphorus source is one of dipotassium hydrogen phosphate, diammonium hydrogen phosphate, and disodium hydrogen phosphate; the growth factor is one of amino acids, small molecule peptides, polycyclic aromatic hydrocarbons, and xylitol.

[0028] Even more preferably, the carbon source is tapioca flour or dextrin; the nitrogen source is one of corn steep liquor, soybean cake powder, and peptone; the phosphorus source is dipotassium hydrogen phosphate or diammonium hydrogen phosphate; the growth factor is amino acids or small molecule peptides.

[0029] Preferably, the specific steps for evaluating the utilization conversion rate of exogenous hydrocarbonophilic emulsifying bacteria to the activator system in step (5) are as follows: Add 200 mL of a 1:1 mixture of target reservoir injection water and formation water and different activator systems to a 500 mL culture flask, sterilize it, then add 5 - 10 mL of hydrocarbonophilic emulsifying bacteria fermentation broth for activation culture, place it in a static culture at the target reservoir temperature for 5 - 10 d, take samples and centrifuge to prepare a bacterial suspension with a concentration of 20 g / L for detecting the dehydrogenase activity. The dehydrogenase acts to reduce the hydrogen acceptor triphenyltetrazolium chloride to triphenylformazan, showing a red color. Use an enzyme-labeling instrument to measure its absorbance at 485 nm, which is the dehydrogenase activity; the higher the dehydrogenase activity, the faster the color development time, and the stronger its degradation and utilization ability to the activator system.

[0030] Preferably, the specific steps for simulating the target reservoir environment and evaluating the oil displacement effect after introducing exogenous hydrocarbonophilic emulsifying bacteria and the activator system in step (6) are as follows: Pack a sandstone core; evacuate and saturate it with formation water, and calculate the pore volume (PV); saturate the dehydrated and degassed crude oil in the target reservoir until the water content of the effluent at the core outlet is the same as that of the target reservoir, and calculate the original oil content of the core; conduct a primary waterflood until 3 PV of formation water is injected, and calculate the primary waterflood recovery rate. Then, culture the pure endogenous bacteria core / exogenous hydrocarbonophilic emulsifying bacteria-introduced core for 20 - 50 d respectively, conduct a secondary waterflood until 3 PV is reached, calculate the enhanced oil recovery of the pure endogenous bacteria core and the exogenous hydrocarbonophilic emulsifying bacteria-introduced core respectively, and screen the exogenous hydrocarbonophilic emulsifying bacteria suitable for the target reservoir according to the increase amplitude △L of the enhanced oil recovery after introducing the exogenous hydrocarbonophilic emulsifying bacteria compared with the pure endogenous bacteria.

[0031] Preferably, the specific evaluation rules for the screening and oil displacement evaluation method of exogenous hydrocarbonophilic emulsifying bacteria in step (6) are shown in the following table:

[0032]

[0033]

[0034] Calculate the comprehensive score of the exogenous hydrocarbonophilic emulsifying bacteria according to the above table. A comprehensive score of 80 or above belongs to category ⅰ, 70 - 79 belongs to category ⅱ, 60 - 69 belongs to category ⅲ, and below 60 belongs to category ⅳ. Among them, categories ⅰ - ⅲ can be applied on-site, and category ⅳ cannot be applied on-site.

[0035] The hydrocarbonophilic emulsifying bacteria of the present invention are an important type of oil-displacing functional bacteria existing in the reservoir environment. On the one hand, they can utilize crude oil as a nutrient to grow and emulsify and strip the crude oil. On the other hand, they can degrade and utilize the activator system secreted by themselves to convert it into beneficial metabolites for oil displacement, thereby improving the emulsification of hydrocarbonophilic crude oil. The selected hydrocarbonophilic emulsifying bacteria play an enhanced oil-displacing role in the reservoir environment and need to have the following characteristics: (1) excellent function of emulsifying and stripping crude oil by hydrocarbonophilic; (2) adapting to the reservoir environment and having good compatibility with endogenous bacteria; (3) high concentration of hydrocarbonophilic emulsifying bacteria and being able to maintain the growth stationary phase for a long time; (4) high dehydrogenase activity for converting the nutrient system into a biological emulsifier; (5) compared with the oil-displacing effect of pure endogenous bacteria, it can further improve the crude oil recovery rate.

[0036] The screening and evaluation of the hydrocarbonophilic emulsifying bacteria of the present invention is to first isolate and obtain hydrocarbonophilic emulsifying bacteria with the potential to adapt to the reservoir environment from the injected water and produced fluid in the reservoir environment; then, according to the characteristics of the bacteria growing and emulsifying and stripping crude oil using crude oil as a nutrient, evaluate the growth and emulsifying and stripping crude oil characteristics of the bacteria according to the ratio of the diameter of the crude oil spreading circle to the diameter of the colony under different crude oil viscosity conditions of the hydrocarbonophilic emulsifying bacteria; at the same time, according to the population proportion and emulsification gene copy number index of the hydrocarbonophilic emulsifying bacteria after being introduced into the reservoir environment for cultivation, determine the compatibility between the exogenous hydrocarbonophilic emulsifying bacteria and the reservoir endogenous bacteria.

[0037] The cultivation and evaluation of the activator system of the present invention is that it can first activate the hydrocarbonophilic emulsifying bacteria to produce a high density of bacterial cells, and at the same time can maintain the growth stationary phase of the bacterial cells for a long time to play a long-term oil-displacing role; then, the dehydrogenase secreted by the hydrocarbonophilic emulsifying bacteria can decompose and convert the activator nutrient system into a biological emulsifier, and the utilization conversion rate of the activator nutrient system can be determined by measuring the dehydrogenase activity; finally, an oil-displacing experiment evaluation is carried out by simulating the target reservoir environment. According to the increase in the crude oil recovery rate after the hydrocarbonophilic emulsifying bacteria are introduced into the core compared with the pure endogenous bacteria core, the exogenous hydrocarbonophilic emulsifying bacteria with enhanced oil-displacing effect are screened. Based on the above evaluation indexes, a screening and evaluation method for exogenous hydrocarbonophilic emulsifying bacteria is constructed, and the comprehensive score of the exogenous hydrocarbonophilic emulsifying bacteria is calculated. The comprehensive score of more than 80 points belongs to class I, 70-79 points belongs to class II, 60-69 points belongs to class III, and less than 60 points belongs to class IV. Among them, classes I-III can be applied to the field, and class IV cannot be applied to the field.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] (1) Comprehensively considering the reservoir environmental factors and the influence of the activator system on the growth and metabolism of exogenous hydrocarbonophilic emulsifying bacteria, it ensures that the selected exogenous hydrocarbonophilic emulsifying bacteria can not only adapt to the reservoir environment, but also can be efficiently cultivated and propagated through a suitable activator system, saving costs and broadening the reservoir range;

[0040] (2) For the first time, co-hydrocarbon-utilizing emulsifying bacteria were isolated and cultured from oil reservoir injection water and produced fluid samples, and based on the ratio of the diameter of the oil expansion circle to the diameter of the strain in different crude oil plates, the growth and reproduction of hydrocarbon-utilizing emulsifying bacteria and their ability to emulsify crude oil were quickly judged, thus realizing the screening of oil reservoirs with different physical properties of crude oil;

[0041] (3) The present invention can select hydrocarbon-utilizing emulsifying bacteria with good compatibility with endogenous bacteria in the target oil reservoir environment by using indicators such as the proportion of hydrocarbon-utilizing emulsifying bacteria population and the gene copy number of emulsifying bacteria, improving the quantity and proportion of hydrocarbon-utilizing emulsifying bacteria in the actual oil reservoir ecosystem, making up for the lack of endogenous hydrocarbon-utilizing emulsifying functional bacteria in the oil reservoir environment, and breaking the traditional technical boundary between endogenous and exogenous bacteria;

[0042] (4) The present invention is a method for screening and evaluating hydrocarbon-utilizing emulsifying bacteria in microbial enhanced oil recovery technology, which has the advantages of being systematic, comprehensive, easy to operate and highly targeted, filling the blank of the screening method for hydrocarbon-utilizing emulsifying bacteria with strong compatibility with oil reservoir endogenous bacteria, giving full play to the enhanced oil displacement effect of exogenous bacteria, providing an effective basis for evaluating the field application effect of microbial oil recovery technology, realizing the enhanced oil displacement effect of exogenous hydrocarbon-utilizing emulsifying bacteria, greatly improving the microbial enhanced oil recovery efficiency, and the annual oil increment in the field test using the present invention reaches more than 2000t.

[0043] Specific implementation method

[0044] The present invention will be further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only for illustrating the present invention and do not limit the scope of the present invention in any way.

[0045] Example 1:

[0046] Oil reservoir for screening hydrocarbon-utilizing emulsifying bacteria: Block A of an oil production plant in Shengli Oilfield, with an oil reservoir temperature of 50 °C, a permeability of 520×10 -3 μm 2 , a formation water salinity of 6500 mg / L, a ground crude oil viscosity of 820 mPa·s, and rich in hydrocarbon-utilizing emulsifying bacteria.

[0047] Target oil reservoir for introducing hydrocarbon-utilizing emulsifying bacteria: Block B of an oil production plant in Shengli Oilfield, with an oil reservoir temperature of 56 °C, a permeability of 1500×10 -3 μm 2 , a formation water salinity of 12600 mg / L, a ground crude oil viscosity of 1390 mPa·s, a water cut of 91%, and a lack of hydrocarbon-utilizing emulsifying bacteria.

[0048] (1) Preliminary culture and screening of exogenous hydrocarbon-utilizing emulsifying bacteria

[0049] Take 1 L of water samples from the injection well A-1 and the two production wells A1-1 and A1-2 in Block A. After centrifugation, prepare a bacterial suspension, take samples and coat them on an LB medium for strain isolation and purification, and conduct 16S rDNA molecular biology identification. The specific results are shown in Table 1. It can be seen from this that 4 strains of bacteria are isolated from the injection well A-1, and 7 strains of bacteria are isolated from the production wells A1-1 and A1-2. The common microbial strains are Bacillus subtilis and Bacillus licheniformis, which have the potential to survive strongly in the reservoir environment.

[0050] Table 1 Types of strains and common strains in injected water and produced fluid

[0051]

[0052] Then inoculate the common strains into a fermentation nutrient system containing 0.2 wt% sucrose, 0.2 wt% peptone, 0.1 wt% K 2 HPO 4 and 0.05 wt% yeast powder, and culture at 56 °C for 2 days. The diesel emulsification index EI of the fermentation broth is shown in Table 2. It can be seen from this that the EIs of Bacillus subtilis and Bacillus licheniformis are 95% and 90% respectively. Bacillus subtilis belongs to Class A and scores 4.5, while Bacillus licheniformis belongs to Class B and scores 3.5. 24 respectively 95% and 90%, and Bacillus subtilis belongs to Class A with a score of 4.5, and Bacillus licheniformis belongs to Class B with a score of 3.5. 24 respectively are 95% and 90%, Bacillus subtilis belongs to Class A and scores 4.5, Bacillus licheniformis belongs to Class B and scores 3.5.

[0053] Table 2 Emulsification index of common strains

[0054] Strain <![CDATA[EI 24 (%)]]> Score Bacillus subtilis 95 4.5 Bacillus licheniformis 90 3.5

[0055] (2) Evaluate the growth and emulsification ability of hydrocarbon-utilizing emulsifying bacteria in the target reservoir environment

[0056] Inoculate the screened hydrocarbon-utilizing emulsifying bacteria Bacillus subtilis and Bacillus licheniformis onto a crude oil plate of the target reservoir block B containing 2.0 wt% urea, 1.0 wt% NH 4 NO 3 1.0 wt%, 0.3 wt% KH 2 PO 4 0.3 wt%, K 2 HPO 40.3 wt%, yeast powder 0.1 wt%, agar powder 15 wt%, cultured at 56 °C for 5 days. Both strains of hydrocarbon-emulsifying bacteria can utilize crude oil as nutrients to grow to varying degrees and can effectively produce biosurfactants to emulsify and strip crude oil to produce an oil expansion circle. The specific conditions of the crude oil expansion circle diameter (D) and colony diameter (d) are shown in Table 3. It can be seen from this that since the viscosity of the crude oil in target reservoir block B is 1390 mPa·s, which belongs to type II crude oil, the D / d of Bacillus subtilis is 6.07, belonging to category A with a score of 18, and the D / d of Bacillus licheniformis is 2.77, belonging to category C with a score of 10.

[0057] Table 3 Screening of the growth and emulsifying ability of hydrocarbon-emulsifying bacteria under the condition of crude oil as nutrients

[0058]

[0059] (3) Combine the screening of biological factor effects to select hydrocarbon-emulsifying bacteria compatible with endogenous bacteria in the target reservoir environment

[0060] Add 200 mL of a 1:1 mixture of target reservoir injection water and formation water and a fermentation nutrient system of sucrose 0.2 wt%, peptone 0.2 wt%, K 2 HPO 4 0.1 wt% and yeast powder 0.05 wt% to a 500 mL culture flask, and add 10 mL of the fermentation broth of Bacillus subtilis and Bacillus licheniformis respectively. Culture at 56 °C for 20 days, and analyze the population proportion and emulsification gene copy number of exogenous hydrocarbon-emulsifying bacteria in the activated reservoir ecosystem, as shown in Table 4. After the introduction of exogenous hydrocarbon-emulsifying functional bacteria into the reservoir environment, they interacted with endogenous bacteria. Among them, the population proportion of Bacillus subtilis was 38%, scoring 10.5, and the lg emulsification gene copy number was 8, scoring 7; the population proportion of Bacillus licheniformis was 15%, scoring 17.5, and the lg emulsification gene copy number was 7, scoring 9.

[0061] Table 4 Proportion of hydrocarbon-emulsifying bacteria and emulsification gene copy number in the presence of endogenous bacteria

[0062]

[0063]

[0064] (4) Study the activation growth of hydrocarbon-emulsifying bacteria by the activator system

[0065] Add 1000 mL of a 1:1 mixture of target reservoir injection water and formation water and an activator system (including 0.2 wt% amylose, 0.1 wt% urea, 0.05 wt% dipotassium hydrogen phosphate, and 0.1 wt% amino acid) into a 2000 mL culture flask, sterilize it, then add 5 mL of Bacillus subtilis and Bacillus licheniformis fermentation broth respectively. Incubate one flask at 56 °C for 3 days, take samples, centrifuge, discard the supernatant, keep the thalli, dry them, and measure the thalli weight. The dry weights of Bacillus subtilis and Bacillus licheniformis thalli both score 7. Incubate one flask at 56 °C for 60 days, take samples and test the peak OD of Bacillus subtilis 600nm is 2.0, the duration of the growth stationary phase is 35 days, and the score is 13.5; the peak OD of Bacillus licheniformis 600nm is 1.2, the duration of the growth stationary phase is 20 days, and the score is 10.5, as shown in Table 5

[0066] Table 5 Evaluation of the dry weights of hydrocarbon - emulsifying bacteria thalli and their growth stationary phases

[0067]

[0068] (5) Evaluate the utilization conversion rate of exogenous hydrocarbon - emulsifying bacteria to the activator system

[0069] Add 200 mL of a 1:1 mixture of target reservoir injection water and formation water and different activator systems into a 500 mL culture flask, sterilize it, then add 5 mL of Bacillus subtilis and Bacillus licheniformis fermentation broth respectively. Place it at 56 °C and statically incubate for 5 days. Take samples and centrifuge to prepare a bacterial suspension with a concentration of 20 g / L for dehydrogenase activity detection. The dehydrogenase activities of Bacillus subtilis and Bacillus licheniformis are 8.8 μg / h / mL and 2.6 μg / h / mL respectively, and the scores are shown in Table 6

[0070] Table 6 Evaluation of the dehydrogenase activities of hydrocarbon - emulsifying bacteria

[0071] Strain Dehydrogenase activity (μg / h / mL) Score Bacillus subtilis 8.8 13.5 Bacillus licheniformis 2.6 7.5

[0072] (6) Simulate the target reservoir environment and evaluate the oil displacement effect after introducing exogenous hydrocarbon - emulsifying bacteria and the activator system into the core

[0073] Simulate the conditions of Block B, pack a core with a permeability of 1500×10 -3 μm 2For the core, after the first water flooding until the water cut reached 91%, crude oil displacement experiments were carried out on the pure endogenous bacteria core / exogenous hydrocarbon - degrading emulsifying bacteria - introduced core respectively. The specific displacement effects are shown in Table 7. After introducing Bacillus subtilis into the core, the enhanced oil recovery is 18.4%, the enhanced oil recovery by pure endogenous bacteria is 8.0%, the increase amplitude △L of the enhanced oil recovery compared with pure endogenous bacteria is 10.4%, and the score is 9; the increase amplitude △L of the enhanced oil recovery by Bacillus licheniformis compared with pure endogenous bacteria is 4.0%, and the score is 5.

[0074] Table 7 Enhanced oil recovery by microorganisms

[0075]

[0076]

[0077] According to the above evaluation of the breeding effects of hydrocarbon - degrading emulsifying bacteria Bacillus subtilis and Bacillus licheniformis, the scores are 83 and 58 respectively. Bacillus subtilis belongs to type ⅰ hydrocarbon - degrading emulsifying bacteria, and Bacillus licheniformis belongs to type ⅳ hydrocarbon - degrading emulsifying bacteria and cannot be applied in the field. The specific results are shown in Table 8.

[0078] Table 8 Comprehensive evaluation of the breeding of hydrocarbon - degrading emulsifying bacteria

[0079]

[0080] In Block B of Shengli Oilfield, the technology of enhanced oil recovery by hydrocarbon - degrading emulsifying bacteria was applied. The screened hydrocarbon - degrading emulsifying bacteria Bacillus subtilis and the activator system (starch 0.2wt%, urea 0.1wt%, dipotassium hydrogen phosphate 0.05wt%, amino acid 0.1wt%) were injected into the reservoir in multiple rounds. After implementation, the number and activity of hydrocarbon - degrading emulsifying bacteria in the block were increased, and they interacted effectively with the crude oil in the formation. When the proportion of the hydrocarbon - degrading emulsifying bacteria population in the produced fluid ≤ 20%, exogenous hydrocarbon - degrading emulsifying bacteria were injected again. When the dehydrogenase activity in the produced fluid ≤ 1.0 μg / h / mL, the activator system was injected again to ensure the effective growth and metabolism of hydrocarbon - degrading emulsifying bacteria in the reservoir. After implementation, the water cut decreased from 91% before implementation to 85%, the effective period was 3 years and 2 months, and the cumulative oil increment was 8750 t.

[0081] Example 2:

[0082] The reservoir for screening hydrocarbon - degrading emulsifying bacteria: Block G of a production plant in Shengli Oilfield, with a reservoir temperature of 46 °C, a permeability of 300×10 -3 μm 2 , a formation water salinity of 27800 mg / L, a ground crude oil viscosity of 1920 mPa·s, and rich endogenous hydrocarbon - degrading emulsifying bacteria.

[0083] Target reservoir for introducing hydrocarbonophilic hydrocarbon emulsifying bacteria: Block F of an oil production plant in Shengli Oilfield, reservoir temperature 40°C, permeability 890×10 -3 μm 2 , formation water salinity 8930 mg / L, ground crude oil viscosity 560 mPa·s, water cut 86%, and the activation effect of indigenous hydrocarbonophilic hydrocarbon emulsifying bacteria is poor.

[0084] (1) Preliminary culture and screening of exogenous hydrocarbonophilic hydrocarbon emulsifying bacteria

[0085] Take 1.5 L of water samples from one injection well G-2 and two production wells F2-1 and F2-2 in Block G, prepare a bacterial suspension after centrifugation, sample and coat on LB medium for strain isolation and purification, and conduct 16S rDNA molecular biology identification. The specific results are shown in Table 9. Five strains of bacteria were isolated from the injection well G-2, and nine strains of bacteria were isolated from the production wells F2-1 and F2-2. The common microbial strains are Bacillus tequilensis and Pseudomonas aeruginosa.

[0086] Table 9 Types of strains and common strains in injection water and produced fluid

[0087]

[0088] Then inoculate the common strains Bacillus tequilensis and Pseudomonas aeruginosa into a fermentation nutrient system of sucrose 0.3 wt%, peptone 0.3 wt%, K 2 HPO 4 0.2 wt% and yeast powder 0.1 wt%, and culture at 40°C for 3 days. The diesel emulsification index EI of the fermentation broth 24 is shown in Table 10. It can be seen from this that both Bacillus tequilensis and Pseudomonas aeruginosa belong to Class B with a score of 3.5.

[0089] Table 10 Emulsification index of common strains

[0090] Strain <![CDATA[EI 24 (%)]]> Score Bacillus tequilensis 88 3.5 Pseudomonas aeruginosa 90 3.5

[0091] (2) Evaluate the growth and emulsification ability of hydrocarbonophilic hydrocarbon emulsifying bacteria in the target reservoir environment

[0092] Inoculate the screened hydrocarbonophilic hydrocarbon emulsifying bacteria Bacillus tequilensis and Pseudomonas aeruginosa on the crude oil plate of the target reservoir Block F with urea 2.5 wt%, NH 4 NO 31.2 wt%, KH 2 PO 4 0.35 wt%, K 2 HPO 4 0.4 wt%, yeast powder 0.12 wt%, agar powder 18 wt%. Cultured at 40 °C for 5 days, both strains of hydrocarbon-emulsifying bacteria can utilize crude oil as nutrition to grow to varying degrees and can effectively produce biosurfactants to emulsify and strip crude oil to produce an oil spreading circle. The specific situation of the oil spreading circle diameter (D) and colony diameter (d) of crude oil is shown in Table 11. It can be seen from this that since the viscosity of the crude oil in the target reservoir block G is 560 mPa·s, which belongs to type I crude oil, the D / d of Bacillus subtilis is 7.07, belonging to class B with a score of 14, and the D / d of Bacillus licheniformis is 9.40, belonging to class A with a score of 18.

[0093] Table 11 Screening of the growth and emulsifying ability of hydrocarbon-emulsifying bacteria under the condition of crude oil as nutrition

[0094]

[0095] (3) Combine the screening of biological factor effects to select hydrocarbon-emulsifying bacteria compatible with indigenous bacteria in the target reservoir environment

[0096] Add 200 mL of the mixture of target reservoir injection water and formation water (1:1) and the fermentation nutrition system of sucrose 0.3 wt%, peptone 0.3 wt%, K 2 HPO 4 0.2 wt% and yeast powder 0.1 wt% into a 500 mL culture flask, and introduce 15 mL of the fermentation broth of Bacillus tequilensis and Pseudomonas aeruginosa respectively. Cultivate at 40 °C for 25 days, and analyze the population proportion of exogenous hydrocarbon-emulsifying bacteria and the index of the copy number of emulsifying genes in the activated reservoir ecosystem, as shown in Table 12. After the two strains of hydrocarbon-emulsifying bacteria were introduced into the reservoir environment, they interacted with the indigenous bacteria. The population proportion of Bacillus tequilensis was 32% with a score of 10.5, and the lg copy number of emulsifying genes was 8 with a score of 7; the population proportion of Pseudomonas aeruginosa was 18% with a score of 7.5, and the lg copy number of emulsifying genes was 7 with a score of 7.

[0097] Table 12 Proportion of hydrocarbon-emulsifying bacteria and copy number of emulsifying genes in the presence of indigenous bacteria

[0098]

[0099] (4) Study the activation and growth of hydrocarbon-emulsifying bacteria by the activator system

[0100] Add 1000 mL of a 1:1 mixture of target reservoir injection water and formation water and an activator system consisting of 0.3 wt% tapioca starch, 0.3 wt% peptone, 0.1 wt% disodium hydrogen phosphate, and 0.2 wt% polycyclic aromatic hydrocarbons into a 2000 mL culture flask, sterilize it, and then add 10 mL of Bacillus tequilensis and Pseudomonas aeruginosa fermentation broth respectively. Incubate one flask at 40 °C for 5 days, take samples, centrifuge, discard the supernatant, keep the bacteria, dry them, and weigh the bacteria for detection. The dry weights of Bacillus tequilensis and Pseudomonas aeruginosa bacteria are shown in Table 13. Incubate one flask at 40 °C for 50 days, take samples and test the peak OD of Bacillus tequilensis 600nm is 2.9, the maintenance time of the growth stable period is 26 days, and the score is 10.5; the peak OD of Pseudomonas aeruginosa 600nm is 2.0, the maintenance time of the growth stable period is 8 days, and the score is 7.5, as shown in Table 13.

[0101] Table 13 Evaluation of the dry weight of hydrocarbon-utilizing emulsifying bacteria and the growth stable period

[0102]

[0103] (5) Evaluate the utilization conversion rate of exogenous hydrocarbon-utilizing emulsifying bacteria to the activator system

[0104] Add 200 mL of a 1:1 mixture of target reservoir injection water and formation water and different activator systems into a 500 mL culture flask, sterilize it, and then add 8 mL of Bacillus tequilensis and Pseudomonas aeruginosa fermentation broth respectively. Place it in a static incubator at 40 °C for 8 days, take samples and centrifuge to prepare a bacterial suspension with a concentration of 20 g / L for dehydrogenase activity detection. The dehydrogenase activities of Bacillus tequilensis and Pseudomonas aeruginosa are 9.3 μg / h / mL and 5.9 μg / h / mL respectively, and the scores are shown in Table 14.

[0105] Table 14 Evaluation of the dehydrogenase activity of hydrocarbon-utilizing emulsifying bacteria

[0106] Strain Dehydrogenase activity (μg / h / mL) Score Bacillus tequilensis 9.3 13.5 Pseudomonas aeruginosa 5.9 10.5

[0107] (6) Simulate the target reservoir environment and evaluate the oil displacement effect after introducing exogenous hydrocarbon-utilizing emulsifying bacteria and the activator system into the core

[0108] Simulate the conditions of Block F, fill a core with a permeability of 890×10 -3 μm2 For the core, after the first water flooding until the water cut reaches 86%, crude oil displacement experiments were carried out on the pure endogenous bacteria core / the core with the introduction of exogenous hydrocarbonophilic emulsifying bacteria respectively. The specific displacement effects are shown in Table 15. The enhanced oil recovery by pure endogenous bacteria is 7.6%. After the introduction of Bacillus tequilensis into the core, the enhanced oil recovery is 16.2%. The increase amplitude △L of the enhanced oil recovery compared with that of pure endogenous bacteria is 8.6%, and the score is 7. After the introduction of Pseudomonas aeruginosa into the core, the enhanced oil recovery is 21.4%. The increase amplitude △L of the enhanced oil recovery compared with that of pure endogenous bacteria is 13.8%, and the score is 9.

[0109] Table 15 Enhanced oil recovery by microorganisms

[0110]

[0111] According to the above evaluation, the total scores of the effects of hydrocarbonophilic emulsifying bacteria Bacillus tequilensis and Pseudomonas aeruginosa and the activator system are 74 points and 68 points respectively. Bacillus tequilensis belongs to class II hydrocarbonophilic emulsifying bacteria, and Pseudomonas aeruginosa belongs to class III hydrocarbonophilic emulsifying bacteria. The specific results are shown in Table 16.

[0112] Table 16 Comprehensive evaluation of hydrocarbonophilic emulsifying bacteria

[0113]

[0114]

[0115] In a certain oil production plant block F in Shengli Oilfield, the application of microbial enhanced oil recovery technology is restricted due to the poor activation effect of hydrocarbonophilic emulsifying bacteria. For this block, the short-board hydrocarbonophilic emulsifying bacteria Bacillus tequilensis, Pseudomonas aeruginosa and the activator system of tapioca starch 0.3wt%, peptone 0.3wt%, disodium hydrogen phosphate 0.1wt%, polycyclic aromatic hydrocarbons 0.2wt% were supplemented for the on-site application of hydrocarbonophilic emulsifying bacteria enhanced oil recovery technology. When the proportion of the hydrocarbonophilic emulsifying bacteria population in the produced fluid ≤ 20%, exogenous hydrocarbonophilic emulsifying bacteria were injected again. When the dehydrogenase activity in the produced fluid ≤ 1.0 μg / h / mL, the activator system was injected again to ensure the effective growth and metabolism of hydrocarbonophilic emulsifying bacteria in the reservoir. After implementation, the hydrocarbonophilic emulsifying bacteria in the block reproduced and metabolized, achieving a significant effect of reducing water cut and increasing oil production. The water cut decreased from 86% before implementation to 70%, the effective period was 3 years, and the cumulative oil increase was 7050t.

[0116] Example 3:

[0117] Reservoirs for screening hydrocarbon-utilizing emulsifying bacteria: Block H of an oil production plant in Shengli Oilfield, with a reservoir temperature of 66 °C, a permeability of 1300×10 -3 μm 2 , a formation water salinity of 7800 mg / L, a ground crude oil viscosity of 2620 mPa·s, and abundant hydrocarbon-utilizing emulsifying bacteria.

[0118] Target reservoirs for introducing hydrocarbon-utilizing emulsifying bacteria: Block W of an oil production plant in Shengli Oilfield, with a reservoir temperature of 70 °C, a permeability of 1690×10 -3 μm 2 , a formation water salinity of 38200 mg / L, a ground crude oil viscosity of 15860 mPa·s, a water cut of 90%, and scarce hydrocarbon-utilizing emulsifying bacteria.

[0119] (1) Preliminary culture and screening of exogenous hydrocarbon-utilizing emulsifying bacteria

[0120] Take 1.5 L of water samples from one injection well H-2 and two production wells W3-1 and F3-2 in Block H, prepare a bacterial suspension after centrifugation, sample and coat on an LB medium for strain isolation and purification, and conduct 16S rDNA molecular biology identification. The specific results are shown in Table 17. Four strains of bacteria were isolated from the injection well H-2, and five strains of bacteria were isolated from the production wells H2-1 and H2-2. The common microbial strain is Geobacillus stearothermophilus.

[0121] Table 17 Types of strains and common strains in injection water and produced fluid

[0122]

[0123] Then inoculate the common strain Geobacillus stearothermophilus into a fermentation nutrient system containing 0.25 wt% sucrose, 0.25 wt% peptone, 0.18 wt% K 2 HPO 4 and 0.06 wt% yeast powder and culture at 70 °C for 2.5 d. The diesel emulsification index EI of the fermentation broth is shown in Table 18. It can be seen from this that the EI of Geobacillus stearothermophilus 24 is 95% respectively, belonging to Class A and scoring 5. 24

[0124] Table 18 Emulsification index of common strains

[0125] Strain <![CDATA[EI 24 (%)]]> Score Geobacillus stearothermophilus 100 5

[0126] (2) Evaluate the growth and emulsification ability of hydrocarbon-utilizing emulsifying bacteria in the target reservoir environment

[0127] The screened hydrocarbonophilic emulsifying bacterium Geobacillus stearothermophilus was inoculated on a crude oil plate with 3.0 wt% urea, NH 4 NO 3 2.0 wt%, KH 2 PO 4 0.3 wt%, K 2 HPO 4 0.5 wt%, 0.2 wt% yeast powder, 20 wt% agar powder, and cultured at 70 °C for 4 days. This bacterium can utilize crude oil as a nutrient to grow to varying degrees and can effectively produce biosurfactants to emulsify and strip crude oil to produce an oil expansion circle. The specific situation of the oil expansion circle diameter (D) and colony diameter (d) of the crude oil is shown in Table 19. It can be seen from this that since the viscosity of the crude oil in target reservoir block W is 15,860 mPa·s, which belongs to Class IV crude oil, the D / d of Geobacillus stearothermophilus is 1.3, belonging to Class C with a score of 10.

[0128] Table 19 Screening of the growth and emulsifying ability of hydrocarbonophilic emulsifying bacteria under the condition of using crude oil as a nutrient

[0129]

[0130] (3) Combine the screening of biological factor effects to select hydrocarbonophilic emulsifying bacteria compatible with endogenous bacteria in the target reservoir environment

[0131] Add 200 mL of a 1:1 mixture of target reservoir injection water and formation water and an activator system to a 500 mL culture flask, and introduce 6 mL of Geobacillus stearothermophilus culture solution respectively. Cultivate at 70 °C for 30 days, and analyze the population proportion of exogenous hydrocarbonophilic emulsifying bacteria and the index of the copy number of emulsifying genes in the activated reservoir ecosystem, as shown in Table 20. After the hydrocarbonophilic emulsifying bacteria were introduced into the reservoir environment, they interacted with the endogenous bacteria. The population proportion of Geobacillus stearothermophilus was 56%, scoring 13.5, and the lg copy number of emulsifying genes was 9, scoring 7.

[0132] Table 20 Proportion of hydrocarbonophilic emulsifying bacteria and copy number of emulsifying genes in the presence of endogenous bacteria

[0133]

[0134] (4) Study the activation growth of the activator system on hydrocarbonophilic emulsifying bacteria

[0135] Add 1000 mL of a 1:1 mixture of the target reservoir injection water and formation water and an activator system (including 0.4 wt% dextrin, 0.15 wt% sodium nitrate, 0.08 wt% diammonium hydrogen phosphate, and 0.15 wt% xylitol) into a 2000 mL culture flask, sterilize it, and then add 8 mL of Geobacillus stearothermophilus fermentation broth. Incubate one flask at 70 °C for 4 days, take samples, centrifuge, discard the supernatant, dry the cells, and weigh the cells for detection. The dry weight score of Geobacillus stearothermophilus cells is 9. Incubate another flask at 56 °C for 50 days, take samples and measure the peak OD 600nm of the bacteria to be 3.2, and the growth stable period lasts for 38 days with a score of 13.5, as shown in Table 21 specifically.

[0136] Table 21 Evaluation of the dry weight of hydrocarbon-emulsifying bacteria cells and the growth stable period

[0137]

[0138] (5) Evaluate the utilization conversion rate of exogenous hydrocarbon-emulsifying bacteria to the activator system

[0139] Add 200 mL of a 1:1 mixture of the target reservoir injection water and formation water and different activator systems into a 500 mL culture flask, sterilize it, and then add 5 mL of Geobacillus stearothermophilus fermentation broth respectively. Place it in a static state at 70 °C for 5 days, take samples, centrifuge to prepare a bacterial suspension with a concentration of 20 g / L for dehydrogenase activity detection. The dehydrogenase activities of the bacteria are 9.0 μg / h / mL and 2.6 μg / h / mL, and the scores are shown in Table 22.

[0140] Table 22 Evaluation of the dehydrogenase activity of hydrocarbon-emulsifying bacteria

[0141]

[0142] (6) Simulate the target reservoir environment and evaluate the oil displacement effect after introducing exogenous hydrocarbon-emulsifying bacteria and the activator system into the core

[0143] Simulate the conditions of Block W, pack a core with a permeability of 1690×10 -3 μm 2 Perform crude oil displacement experiments on the pure endogenous bacteria core / the core with exogenous hydrocarbon-emulsifying bacteria introduced respectively after one-time water flooding to a water cut of 90%. The specific displacement effects are shown in Table 23. Among them, the enhanced oil recovery by pure endogenous bacteria is 9.6%, and the enhanced oil recovery after introducing Geobacillus stearothermophilus into the core is 23.8%. The increase amplitude △L of the enhanced oil recovery compared with pure endogenous bacteria is 14.2%, with a score of 9.

[0144] Table 23 Microbial enhanced oil recovery

[0145]

[0146]

[0147] According to the above evaluation of the hydrocarbon - emulsifying bacterium Geobacillus stearothermophilus and the activator system, the total score is 80.5, belonging to Class I hydrocarbon - emulsifying bacteria. The specific results are shown in Table 24.

[0148] Table 24 Comprehensive evaluation of hydrocarbon - emulsifying bacteria

[0149]

[0150] In a certain oil production plant block W in Shengli Oilfield, the application of microbial enhanced oil recovery technology is restricted due to the lack of hydrocarbon - emulsifying bacteria. The hydrocarbon - emulsifying bacterium Geobacillus stearothermophilus and the activator system of dextrin 0.4wt%, sodium nitrate 0.15wt%, diammonium hydrogen phosphate 0.08wt%, and xylitol 0.15wt% are injected into the reservoir in multiple rounds. When the proportion of the hydrocarbon - emulsifying bacteria population in the produced fluid ≤ 20%, exogenous hydrocarbon - emulsifying bacteria are injected again. When the dehydrogenase activity in the produced fluid ≤ 1.0 μg / h / mL, the activator system is injected again to ensure the quantity and activity of hydrocarbon - emulsifying bacteria in the reservoir. After implementation, the water cut is reduced from 90% before implementation to 83%, the effective period is 2 years and 6 months, and the cumulative oil increment is 6880t.

[0151] Example 4:

[0152] The reservoir for screening hydrocarbon - emulsifying bacteria: Block S in a certain oil production plant of Shengli Oilfield, reservoir temperature 80°C, permeability 1600×10 -3 μm 2 , formation water salinity 2200mg / L, ground crude oil viscosity 3100mPa·s, rich in hydrocarbon - emulsifying bacteria.

[0153] The target reservoir for introducing hydrocarbon - emulsifying bacteria: Block N in a certain oil production plant of Shengli Oilfield, reservoir temperature 66°C, permeability 980×10 -3 μm 2 , formation water salinity 7360mg / L, ground crude oil viscosity 5730mPa·s, water cut 95%, and it is difficult to activate endogenous hydrocarbon - emulsifying bacteria.

[0154] (1) Preliminary culture and screening of exogenous hydrocarbon - emulsifying bacteria

[0155] Take 2.0 L of water samples from the 1 water injection well S-3 and 2 oil wells N3-1 and N3-2 in block S. After centrifugation, prepare a bacterial suspension, sample and coat it on an LB medium for strain isolation and purification, and conduct 16S rDNA molecular biology identification. The specific results are shown in Table 25. 3 strains of bacteria were isolated from the water injection well S-3, and 6 strains of bacteria were isolated from the oil wells N3-1 and N3-2. The common microbial strain is Thermoleophilum album.

[0156] Table 25 Types of strains and common strains in injected water and produced fluid

[0157]

[0158]

[0159] Then inoculate the common strain Thermoleophilum album into a fermentation nutrient system: sucrose 0.2 wt%, peptone 0.2 wt%, K 2 HPO 4 0.1 wt% and yeast powder 0.05 wt%. Culture at 66 °C for 3 d. The diesel emulsification index EI of the fermentation broth 24 is shown in Table 26. It can be seen from this that the emulsification index of Thermoleophilum album is 96%, belonging to Class A with a score of 5.

[0160] Table 26 Emulsification index of common strains

[0161] Strain <![CDATA[EI 24 (%)]]> Score Thermoleophilum album 96 5

[0162] (2) Evaluate the growth and emulsification ability of hydrocarbonophilic emulsifying bacteria in the target reservoir environment

[0163] Inoculate the screened hydrocarbonophilic emulsifying bacterium Thermoleophilum album on a crude oil plate of the target reservoir block N: urea 2.0 wt%, NH 4 NO 3 1.0 wt%, KH 2 PO 4 0.35 wt%, K 2 HPO 4 0.4 wt%, yeast powder 0.1 wt%, agar powder 20 wt%. Culture at 66 °C for 5 d. The hydrocarbonophilic emulsifying bacterium can utilize crude oil as a nutrient for growth and can effectively produce biosurfactants to emulsify and strip the crude oil to produce an oil expansion circle. The specific situation of the diameter (D) of the crude oil expansion circle and the diameter (d) of the colony is shown in Table 27. It can be seen from this that since the viscosity of the crude oil in the target reservoir block N is 5730 mPa·s, belonging to Class III crude oil, the D / d of Thermoleophilum album is 7.07, belonging to Class B with a score of 14.

[0164] Screening of the growth and emulsifying ability of hydrocarbon - degrading emulsifying bacteria under the condition of crude oil as nutrition

[0165]

[0166] (3) Combine the screening affected by biological factors to select hydrocarbon - degrading emulsifying bacteria compatible with indigenous bacteria in the target reservoir environment

[0167] Add 200 mL of the mixture of target reservoir injection water and formation water (1:1) and the fermentation nutrient system (sucrose 0.3 wt%, peptone 0.2 wt%, K 2 HPO 4 0.2 wt% and yeast powder 0.1 wt%) into a 500 - mL culture flask, and introduce 10 mL of Thermoleophilum album culture solution. Cultivate at 66 °C for 30 d, and analyze the population proportion of exogenous hydrocarbon - degrading emulsifying bacteria and the index of emulsification gene copy number in the activated reservoir ecosystem. As shown in Table 28. After the hydrocarbon - degrading emulsifying bacteria are introduced into the reservoir environment, they interact with indigenous bacteria. The population proportion of Thermoleophilum album is 45% with a score of 13.5, and the lg emulsification gene copy number is 6 with a score of 7.

[0168] Table 28 Proportion of hydrocarbon - degrading emulsifying bacteria and emulsification gene copy number in the presence of indigenous bacteria

[0169] Strain Population proportion of hydrocarbon-emulsifying bacteria (%) lg Emulsification gene copy number (copies / mL) Thermoleophilum album 45 6 Score 10.5 7

[0170] (4) Study the activation and growth of hydrocarbon - degrading emulsifying bacteria by the activator system

[0171] Add 1000 mL of the mixture of target reservoir injection water and formation water (1:1) and the activator system (molasses 0.3 wt%, soybean cake powder 0.2 wt%, dipotassium hydrogen phosphate 0.05 wt%, small - molecule peptide 0.1 wt%) into a 2000 - mL culture flask respectively, sterilize, and then add 5 mL of Thermoleophilum album fermentation broth. One flask is cultivated at 56 °C for 3 d, sampled, centrifuged, the supernatant is discarded, and the cells are dried for cell weight detection. The dry weight score of Thermoleophilum album cells is 8.6. One flask is cultivated at 66 °C for 60 d, sampled to test the peak OD 600nm of Bacillus subtilis is 1.1, and the maintenance time of the growth stable period is 40 d with a score of 13.5, as shown in Table 29.

[0172] Table 29 Evaluation of the dry weight of hydrocarbon - degrading emulsifying bacteria cells and the growth stable period

[0173]

[0174] (5) Evaluate the utilization conversion rate of exogenous hydrocarbon-emulsifying bacteria to the activator system

[0175] Add 200 mL of the mixed solution of target reservoir injection water and formation water at a ratio of 1:1 and different activator systems to a 500 mL culture flask, sterilize it, then add 5 mL of Thermoleophilum album fermentation broth respectively, place it in static culture at 66 °C for 5 days, take samples and centrifuge to prepare a bacterial suspension with a concentration of 20 g / L for dehydrogenase activity detection. The dehydrogenase activity of this bacterium is 9.6 μg / h / mL, and the scoring is shown in Table 30.

[0176] Table 30 Evaluation of dehydrogenase activity of hydrocarbon-emulsifying bacteria

[0177]

[0178] (6) Simulate the target reservoir environment and evaluate the oil displacement effect after introducing exogenous hydrocarbon-emulsifying bacteria and activator system into the core

[0179] Simulate the conditions of Block N, pack a core with a permeability of 980×10 -3 μm 2 Perform the crude oil displacement experiment of pure endogenous bacteria core / exogenous hydrocarbon-emulsifying bacteria introduced into the core after water flooding to a water cut of 95% for the first time. The specific displacement effect is shown in Table 31. The oil recovery rate increased by 10.0% with pure endogenous bacteria, and the oil recovery rate increased by 16.0% after Thermoleophilum album was introduced into the core. The increase amplitude △L of the oil recovery rate compared with pure endogenous bacteria is 6.0%, and the score is 7.

[0180] Table 31 Microbial enhanced oil recovery

[0181]

[0182] According to the above evaluation, the total score of the effect of hydrocarbon-emulsifying bacteria Thermoleophilum album and the activator system is 77.5, belonging to type II hydrocarbon-emulsifying bacteria. The specific results are shown in Table 32.

[0183] Table 32 Comprehensive evaluation of hydrocarbon-emulsifying bacteria

[0184]

[0185]

[0186] In Block N of a certain oil production plant in Shengli Oilfield, the application of the microbial enhanced oil recovery technology was restricted due to the lack of hydrocarbon-utilizing emulsifying bacteria. The screened hydrocarbon-utilizing emulsifying bacteria Thermoleophilum album and the activator system consisting of 0.3 wt% molasses, 0.2 wt% soybean cake powder, 0.05 wt% dipotassium hydrogen phosphate, and 0.1 wt% small molecule peptides were used for the application of the hydrocarbon-utilizing emulsifying bacteria enhanced oil recovery technology in Block N. After implementation, the number and activity of hydrocarbon-utilizing emulsifying bacteria in the block were increased, and they effectively interacted with the crude oil in the formation. When the proportion of the hydrocarbon-utilizing emulsifying bacteria population in the produced fluid ≤ 20%, exogenous hydrocarbon-utilizing emulsifying bacteria were injected again. When the dehydrogenase activity in the produced fluid ≤ 1.0 μg / h / mL, the activator system was injected again to ensure the effective growth and metabolism of hydrocarbon-utilizing emulsifying bacteria in the reservoir. After implementation, the hydrocarbon-utilizing emulsifying bacteria in the block reproduced and metabolized, and the number reached 2.2×10 8 cells / mL, effectively interacting with the crude oil in the formation. The water cut decreased from 95% before implementation to 90%, the effective period was 2 years and 6 months, and the cumulative oil increment was 6020 t.

[0187] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including the combination of each technical feature in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A method for screening and evaluating exogenous hydrocarbon - emulsifying functional bacteria, characterized in that, the screening and evaluation method specifically includes the following steps: (1) Preliminary cultivation and screening of exogenous hydrocarbon - emulsifying bacteria; (2) Evaluating the growth and emulsifying ability of hydrocarbon - emulsifying bacteria in the target reservoir environment; (3) Combining the influence of biological factors to screen hydrocarbon - emulsifying bacteria compatible with the endogenous bacteria in the target reservoir; (4) Studying the activation growth of hydrocarbon - emulsifying bacteria by the activator system; (5) Evaluating the utilization conversion rate of the exogenous hydrocarbon - emulsifying bacteria to the activator system; (6) Simulating the target reservoir environment and evaluating the oil displacement effect after introducing the exogenous hydrocarbon - emulsifying bacteria and the activator system into the core; (7) According to the evaluation results of steps (1), (2), (3), (4), (5) and (6), constructing a screening and oil displacement evaluation method for exogenous hydrocarbon - emulsifying bacteria; (8) Field application of exogenous hydrocarbon - emulsifying functional bacteria.

2. The method for screening and evaluating exogenous hydrocarbon - emulsifying functional bacteria according to claim 1, characterized in that, in step (1), the preliminary cultivation and screening of the exogenous hydrocarbon - emulsifying bacteria are carried out as follows: respectively take 1 - 2 L of injection water and produced fluid from the target reservoir into a sampling bucket, centrifuge and concentrate to prepare a bacterial suspension, sample and coat it on an LB medium, carry out strain isolation and purification at the reservoir temperature, extract the genome of the cultured strains, use the bacterial 16S rDNA sequencing method for molecular biological identification of the strains, determine the types of common strains in the injection water and the produced fluid, and the common strains can represent the strains with strong reservoir adaptation potential that survive in the reservoir environment introduced into the injection water; then inoculate the common strains into a fermentation nutrition system respectively for fermentation culture for 2 - 3 d, and measure the emulsification index of the fermentation broth.

3. The method for screening and evaluating exogenous hydrocarbon - emulsifying functional bacteria according to claim 2, characterized in that, The fermentation nutrition system is 0.2 - 0.3 wt% sucrose, 0.2 - 0.3 wt% peptone, 2 K 4 HPO 0.1 - 0.2 wt% and 0.05 - 0.1 wt% yeast powder.

4. The method for screening and evaluating exogenous hydrocarbon - emulsifying functional bacteria according to claim 1, characterized in that, in step (2), the evaluation of the growth and emulsifying ability of hydrocarbon - emulsifying bacteria in the target reservoir environment is carried out as follows: first inoculate the screened hydrocarbon - emulsifying bacteria into a crude oil plate prepared with the crude oil of the target reservoir, and culture it at the target reservoir temperature for 3 - 5 d. The ratio (D / d) of the diameter (D) of the crude oil spreading circle of the hydrocarbon - emulsifying bacteria to the diameter (d) of the colony can represent the growth and emulsifying ability of the hydrocarbon - emulsifying bacteria. According to the size of the D / d ratio, screen out the hydrocarbon - emulsifying bacteria that can grow with crude oil as the sole carbon source and can effectively emulsify and strip crude oil.

5. The method for screening and evaluating exogenous hydrocarbon - emulsifying functional bacteria according to claim 4, characterized in that, The crude oil plate contains 2.0 - 3.0 wt% of urea, 4 NO 3 1.0 - 2.0 wt%, KH 2 PO 4 0.3 - 0.5 wt%, K 2 HPO 4 0.3 - 0.5 wt%, 0.1 - 0.2 wt% of yeast powder, 15 - 20 wt% of agar powder. Sterilize at 121 °C for 20 min. Take it out and pour the plate. Wait for the plate to solidify, add 10 mL of sterilized crude oil, and spread it evenly with a spreading rod.

6. The method for screening and evaluating exogenous hydrocarbon - emulsifying functional bacteria according to claim 1, characterized in that, In step (3), the hydrocarbonophilic emulsifying bacteria compatible with the indigenous bacteria in the target reservoir are screened by considering the influence of biological factors. The specific steps are as follows: In a 500 mL culture flask, add 200 mL of a 1:1 mixture of the target reservoir injection water and formation water, and the fermentation nutrient system, and add 10 - 20 mL of the hydrocarbonophilic emulsifying bacteria culture solution. Place it in a static state at the temperature of the target reservoir for 20 - 30 d. According to the population proportion of exogenous hydrocarbonophilic emulsifying bacteria and the copy number of emulsification genes in the activated reservoir ecosystem, judge the compatibility between the exogenous hydrocarbonophilic emulsifying bacteria and the indigenous bacteria in the reservoir.

7. The method for screening, breeding and evaluating an exogenous hydrocarbonophilic emulsifying functional bacterium according to claim 1, characterized in that In step (4), the activation and growth of hydrocarbonophilic emulsifying bacteria by the activator system are studied. The specific steps are as follows: In a 2000 mL culture flask, add 1000 mL of a 1:1 mixture of the target reservoir injection water and formation water and the activator system, sterilize it, then add 5 - 10 mL of the hydrocarbonophilic emulsifying bacteria fermentation broth, place it in a culture at the temperature of the target reservoir, and conduct evaluation studies on the dry weight of the bacteria and the growth stationary phase respectively.

8. The method for screening, breeding and evaluating an exogenous hydrocarbonophilic emulsifying functional bacterium according to claim 7, characterized in that The evaluation of the dry weight of the bacteria refers to placing the culture flask in a static state at the temperature of the target reservoir for 3 - 5 d, sampling, centrifuging, discarding the supernatant, leaving the bacteria for drying, and conducting the weighing detection of the bacteria.

9. The method for screening, breeding and evaluating an exogenous hydrocarbonophilic emulsifying functional bacterium according to claim 7, characterized in that The evaluation of the growth stationary phase refers to placing the culture flask in a static state at the temperature of the target reservoir for 60 d, detecting the OD600nm value every 2 d, and counting the maintenance time of the peak OD600nm value of the hydrocarbonophilic emulsifying bacteria.

10. The method for screening, breeding and evaluating an exogenous hydrocarbonophilic emulsifying functional bacterium according to claim 7, characterized in that The activator system refers to 0.2 - 0.5 wt% of carbon source, 0.1 - 0.3 wt% of nitrogen source, 0.05 - 0.1 wt% of phosphorus source, and 0.1 - 0.2 wt% of growth factor.

11. The method for screening, breeding and evaluating an exogenous hydrocarbonophilic emulsifying functional bacterium according to claim 10, characterized in that The carbon source is one of starch, cassava flour, dextrin, molasses; the nitrogen source is one of corn steep liquor, soybean cake powder, peptone, urea, ammonium chloride, sodium nitrate; the phosphorus source is one of dipotassium hydrogen phosphate, diammonium hydrogen phosphate, disodium hydrogen phosphate, and the growth factor is one of amino acids, small molecule peptides, polycyclic aromatic hydrocarbons, xylitol.

12. The method for screening, breeding and evaluating an exogenous hydrocarbonophilic emulsifying functional bacterium according to claim 11, characterized in that The carbon source is cassava flour or dextrin; the nitrogen source is one of corn steep liquor, soybean cake powder, peptone; the phosphorus source is dipotassium hydrogen phosphate or diammonium hydrogen phosphate; the growth factor is amino acids or small molecule peptides.

13. The method for screening, breeding and evaluating an exogenous hydrocarbonophilic emulsifying functional bacterium according to claim 1, characterized in that The specific steps for evaluating the utilization conversion rate of exogenous hydrocarbon-emulsifying bacteria to the activator system in step (5) are as follows: Add 200 mL of a 1:1 mixture of target reservoir injection water and formation water and different activator systems into a 500 mL culture flask, sterilize it, then add 5 - 10 mL of hydrocarbon-emulsifying bacteria fermentation broth for activation culture, place it in a static culture at the target reservoir temperature for 5 - 10 d, take samples and centrifuge to prepare a bacterial suspension with a concentration of 20 g / L for the detection of dehydrogenase activity.

14. The method for screening and evaluating an exogenous hydrocarbon-emulsifying functional bacterium according to claim 1, characterized in that the specific steps for simulating the target reservoir environment in step (6) to evaluate the oil displacement effect after introducing exogenous hydrocarbon-emulsifying bacteria and the activator system are as follows: Pack a sandstone core; evacuate and saturate it with formation water, calculate the pore volume (PV); saturate the dehydrated and degassed crude oil in the target reservoir until the water cut of the effluent at the core outlet is the same as that of the target reservoir, calculate the original oil content of the core; conduct a primary water drive until 3 PV of formation water is injected, calculate the primary water drive recovery rate, then culture the pure endogenous bacteria core / the core with exogenous hydrocarbon-emulsifying bacteria introduced for 20 - 50 d respectively, conduct a secondary water drive until 3 PV is reached, calculate the enhanced oil recovery of the pure endogenous bacteria core and the core with exogenous hydrocarbon-emulsifying bacteria introduced respectively, and screen the exogenous hydrocarbon-emulsifying bacteria suitable for the target reservoir according to the increase amplitude △L of the enhanced oil recovery after introducing exogenous hydrocarbon-emulsifying bacteria compared with that of the pure endogenous bacteria.

15. The method for screening and evaluating an exogenous hydrocarbon-emulsifying functional bacterium according to claim 1, characterized in that the specific evaluation rules for the method for screening and evaluating an exogenous hydrocarbon-emulsifying bacterium in step (6) are shown in the following table: Calculate the comprehensive score of the exogenous hydrocarbon-emulsifying bacteria according to the above table. A comprehensive score of more than 80 points belongs to class ⅰ, 70 - 79 points belongs to class ⅱ, 60 - 69 points belongs to class ⅲ, and less than 60 points belongs to class ⅳ. Among them, classes ⅰ - ⅲ can be applied in the field, and class ⅳ cannot be applied in the field.

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