阅读说明：本技术 一株铁还原菌Tessaracoccus oleiagri DH10菌株及其应用 (Iron reducing bacterium Tessaracococcus oleiagri DH10 strain and application thereof ) 是由 佘跃惠 董浩 孙姗姗 张凡 苏三宝 张涵 翁雪 喻高明 郑安应 李杨 于 2021-07-15 设计创作，主要内容包括：本发明公开了一株铁还原菌Tessaracoccus oleiagri DH10菌株及其应用,所述Tessaracoccus oleiagri DH10菌株已于2021年04月19日保藏于中国典型培养物保藏中心,保藏编号为：CCTCC NO：M 2021404。本发明从油藏环境中分离筛选得到了一株可高效还原Fe(III)的铁还原菌Tessaracoccus oleiagri DH10,扫描电镜分析等表明其可有效分解贫铁蒙脱石矿物,并抑制黏土膨胀；同时岩心实验表明,Tessaracoccus oleiagri DH10生物制剂可降低储层岩心水敏性,降低注水压力,可将其应用于原油采收中,有效提高原油采收率。(The invention discloses an iron reducing bacterium Tessaracoccus oleiagri DH10 strain and application thereof, wherein the Tessaracoccus oleiagri DH10 strain is deposited in China center for type culture Collection at 04-19.2021, and the deposit numbers are as follows: CCTCC NO: m2021404. The invention separates and screens an iron reducing bacterium Tessaracococcus oleiagri DH10 which can efficiently reduce Fe (III) from an oil reservoir environment, and scanning electron microscope analysis and the like show that the iron reducing bacterium Tessaracococcus oleiagri DH10 can effectively decompose lean iron montmorillonite minerals and inhibit clay swelling; meanwhile, core experiments show that the Tessaracoccus oleiagri DH10 biological agent can reduce the water sensitivity of the reservoir core and reduce the water injection pressure, and can be applied to crude oil recovery to effectively improve the crude oil recovery rate.)

1. An iron reducing bacterium Tessaracococcus oleiagri DH10 strain, wherein the preservation number of the Tessaracococcus oleiagri DH10 strain is: CCTCC NO: m2021404.

2. A microbial preparation comprising the strain of iron-reducing bacterium tessaraccus oleiagri DH10 according to claim 1.

3. The microbial preparation of claim 2, wherein the microbial preparation is a solid or liquid preparation.

4. A biological expansion-contraction microbial inoculum is characterized by comprising: a nutrient medium, and the strain of iron-reducing bacteria Tessaracococcus oleiagri DH10 of claim 1 or the microbial preparation of claim 2.

5. The biological turgor agent as claimed in claim 4, wherein the nutrient medium in the biological turgor agent is LB medium; or comprises: 10-50g/L of sucrose, 10-40g/L of sodium acetate, 3-20g/L of sodium lactate and MgSO₄ 0.1-2g/L，KCl 2-18g/L，MnSO₄ 5-9g/L，CuSO₄ 5-10g/L，ZnSO₄ 5-12g/L，KH₂PO₄1-7g/L, 2-10g/L montmorillonite, and 5.0-9.0 pH.

6. The biological puffing agent of claim 4, wherein the strain Tessaracococcus oleiagri DH10 of claim 1 or the microbial preparation of claim 2 is inoculated into a nutrient medium and fermented at pH 5-9.5 and temperature 20-60 ℃ to obtain the biological puffing agent.

7. The iron-reducing bacteria Tessaracococcus oleiagri DH10 strain of claim 1, or the microbial preparation of claim 2, or the use of the biological strain of claim 4 in the reduction of Fe (III).

8. The use of the strain of iron-reducing bacteria Tessaracococcus oleiagri DH10 according to claim 1, or the microbial preparation according to claim 2, or the biological swollenination inoculant according to claim 4 for inhibiting clay swelling.

9. The use of the iron-reducing bacterium Tessaracococcus oleiagri DH10 strain of claim 1, or the microbial preparation of claim 2, or the biological swollenination inoculant of claim 4 for reducing reservoir injection pressure.

10. The use of the iron-reducing bacterium strain Tessaracococcus oleiagri DH10 of claim 1, or the microbial preparation of claim 2, or the biological reswelling agent of claim 4 for enhanced oil recovery.

Technical Field

The invention belongs to the technical field of microbial oil recovery, and particularly relates to an iron reducing bacterium Tessaracococcus oleiagri DH10 strain and application thereof.

Background

With the development of the petroleum industry, the conventional oil and gas resources in China are less and less, the demand of the social oil and gas resources is higher and higher, and the contradiction causes that the external dependence of the petroleum supply in China is continuously maintained at a high level, so that the energy safety is threatened. Oil and gas resources such as low-permeability oil reservoirs and water-sensitive oil reservoirs are break-through points for increasing the oil yield in the next long time, and meanwhile, the method is also necessary to be an important research field. As is well known, the water-sensitive clay mineral is one of the main factors for restricting the development level of low-permeability oil reservoirs and water-sensitive oil reservoirs, so that the stabilization of the water-sensitive clay mineral is the key point for improving the development level of the low-permeability oil reservoirs and the water-sensitive oil reservoirs. Low permeability and water sensitive reservoirs are typically higher in clay mineral content. The clay content determines the permeability of a reservoir, the permeability is greatly changed even if the clay content is slightly changed, when the clay content reaches 35-40%, the reservoir is almost impermeable, the higher the clay mineral content is, the higher the clay swelling rate is and the stronger the cation exchange capacity is, the stronger the water sensitivity of the reservoir is, and the clay mineral type influences the seepage characteristics of the reservoir. Swelling clays are key factors affecting the permeability of low permeability reservoirs, and swelling of bentonite reduces permeability.

Reduction of Fe (III)The functional microorganism is a special microorganism population which can utilize organic matter as an electron donor, oxidize the organic matter and use Fe³⁺As the only electron acceptor, Fe (III) is reduced into Fe (II), and the energy required for self-growth is obtained in the reduction and metabolism process. Metabolites of iron reducing microorganisms can change the physicochemical environment of the mineral surface, and are the main motive force for mineral decomposition. The microbial mineral decomposition rate is several orders of magnitude higher than that of single chemical decomposition, and as shown in the research of Science journal published in 2004 by Kim et al, a dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 can reduce the trivalent iron in ferri-rich montmorillonite and promote the conversion of montmorillonite to illite within a period of two weeks. Daniel et al in 2002 discovered for the first time that thermophilic anaerobic methanogens can also reduce ferric iron in the clay mineral structure, resulting in a phase transition of the clay mineral. These findings break through the long-term understanding that the illite conversion process of montmorillonite is controlled by temperature, pressure and time, and break through the limitation of large time scale of conversion between clay minerals. Therefore, the iron-reducing microorganism in the oil reservoir is utilized to inhibit the hydration expansion or contraction expansion of the clay, improve the seepage environment of crude oil fluid and have important significance for improving the crude oil recovery ratio of the oil reservoir.

In the process of microbial oil recovery, different oil reservoirs have certain requirements on strains, a lot of iron-producing reducing bacteria are reported at present, but few iron-reducing bacteria are separated from the oil reservoirs, and no relevant research report is found on the functional characteristics of the Tessaracocccus microorganisms Tessaracocccus oleiagri separated from the oil reservoir environment and the application of the Tessaracocccus oleiagri in oil exploitation and clay expansion inhibition at present.

Disclosure of Invention

One of the purposes of the invention is to provide an iron reducing bacterium Tessaracococcus oleiagri DH10 strain, wherein the strain is isolated from an oil reservoir environment and is deposited in China center for type culture Collection at 04 and 19 months in 2021, and the addresses are as follows: the preservation number of the Tessaracococcus oleiagri DH10 strain is as follows: CCTCC NO: m2021404.

The invention also aims to provide a microbial preparation which comprises the strain of the iron-reducing bacterium Tessaracococcus oleiagri DH 10.

Further, the microbial preparation is a solid preparation or a liquid preparation.

The invention also aims to provide a biological expansion-contraction microbial inoculum, which comprises the following components in part by weight: a nutrient medium, and the strain of the iron-reducing bacterium Tessaracococcus oleiagri DH10 or the microbial preparation.

Further, the nutrient medium in the biological puffing agent is an LB medium or comprises: 10-50g/L of sucrose, 10-40g/L of sodium acetate, 3-20g/L of sodium lactate and MgSO₄ 0.1-2g/L，KCl 2-18g/L，MnSO₄ 5-9g/L，CuSO₄ 5-10g/L，ZnSO₄ 5-12g/L，KH₂PO₄1-7g/L, 2-10g/L montmorillonite, and 5.0-9.0 pH.

Further, inoculating the strain of the iron reducing bacterium Tessaracococcus oleiagri DH10 or the microbial preparation into a nutrient medium, and fermenting at the conditions of pH 5-9.5 and temperature of 20-60 ℃ to obtain the biological expansion-shrinking bacterial agent.

The fourth purpose of the invention is to provide the application of the strain Tessaracococcus oleiagri DH10, or the microbial preparation, or the biological expansion-contraction microbial inoculum in reducing Fe (III).

The fifth purpose of the invention is to provide the strain Tessaracococcus oleiagri DH10, or the microbial preparation, or the application of the biological swelling-shrinking bacterial agent in inhibiting clay swelling.

The invention also aims to provide the application of the iron reducing bacteria Tessaracocccus oleiagri DH10 strain, or the microbial preparation, or the biological expansion-contraction microbial inoculum in reducing the injection pressure of a low-permeability reservoir.

The seventh purpose of the invention is to provide the application of the iron reducing bacteria Tessaracococcus oleiagri DH10 strain, or the microbial preparation, or the biological expansion-contraction microbial inoculum in improving the recovery ratio of crude oil.

Compared with the prior art, the invention has the beneficial effects that: the reducible Fe is obtained by separating and screening from the oil reservoir environment³⁺The analysis of the iron reducing bacterium Tessaracococcus oleiagri DH10, a scanning electron microscope, XRD and the like shows that the Tessaracococcus oleiagri DH10 strain can effectively decompose and corrode montmorillonite minerals, and Fe in the montmorillonite minerals³⁺Reduction to Fe²⁺Effectively inhibiting the clay expansion; meanwhile, core experiments show that the Tessaracoccus oleiagri DH10 biological agent can reduce the water injection pressure in oil development, and the Tessaracoccus oleiagri DH10 strain is applied to crude oil recovery, so that the recovery rate of crude oil can be effectively improved.

Drawings

FIG. 1 shows inoculation of DH10 strain into Fe (OH) according to example 1 of the present invention₃Color change of the culture medium before and after;

FIG. 2 shows Fe in montmorillonite as compared with that in a blank control group after inoculation of DH10 strain in example 2 of the present invention²⁺The change in concentration over time;

FIG. 3 is a scanning electron microscope photograph of montmorillonite samples before and after the action of DH10 strain in example 2 of the present invention, wherein FIG. 3-A is the original montmorillonite sample, and FIG. 3-B is the montmorillonite sample after the action of strain DH10 for 7 days.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isolation and identification of iron-reducing bacteria

1. Isolation of iron-reducing bacteria

The invention provides an iron reducing bacterium separated from an oil reservoir environment, and a screening method thereof comprises the following steps: according to the conventional strain screening method, 10mL of water sample collected from an oil field is taken and inoculated with 100mL of Fe (OH)₃(0.5％Fe(OH)₃W: v) anaerobic culture at 35 deg.C1 week, selecting an experimental group with obvious solid phase color darkening, taking 100 mu L of fermentation liquor in an anaerobic operation station under anaerobic condition, coating the fermentation liquor on an LB agar plate culture medium, and carrying out anaerobic culture at 35 ℃ for 48 hours; picking single colonies with different forms, streaking and purifying on an LB agar plate culture medium, and culturing for 48h at 35 ℃. Picking single colonies, inoculating by enrichment culture into Fe (OH)₃And (3) standing and anaerobically culturing for 72h at 35 ℃ in a culture medium, and selecting a strain corresponding to an experimental group with obviously darkened solid phase color. In this example, the primary screening revealed that the sample contained microorganisms capable of significantly darkening the color of the solid phase (the color change of the solid phase before and after culturing the bacteria is shown in FIG. 1), indicating that the microorganisms obtained by screening were capable of reducing Fe (III).

In this example, 1 strain capable of reducing fe (iii) was obtained by preliminarily screening a microorganism capable of metabolizing fe (iii) as a unique electron acceptor, further culturing and acclimating the microorganism, and then separating the strain, and the strain was named DH 10. The concentrations of Fe (III) and Fe (II) in the sample are measured by spectrophotometry, and the result shows that the reduction efficiency of the DH10 strain to Fe (III) in the culture medium can reach 35.3 percent within 4 days.

2. Molecular biological identification

According to the 16S rRNA identification method of the conventional strain, genome DNA of the strain DH10 is extracted, a corresponding primer is designed for PCR amplification, an amplification product is detected by agarose gel electrophoresis, and the PCR product is sent to Nanjing Pakino gene technology company Limited for DNA sequencing. Sequencing of PCR amplification products of DH10 NCBI submitted to BLAST searches and homology comparisons determined strain DH10 to be Tessaracococcus oleiagri (99.72% similarity to Tessaracococcus oleiagriola strain SL014B-20A 11). The 16S rRNA sequence of the DH10 strain is aligned with the Genbank database sequence as shown in Table 1.

Alignment of the 16S rRNA sequence of DH10 with Genbank database sequences in Table 1

The strain DH10 of the present invention can be preserved by the following method:

(1) short-term preservation: the strain is streaked on a slant culture medium, cultured for 48 hours at 35 ℃, and then placed at 4 ℃ for short-term storage.

(2) And (3) long-term storage: adopts glycerol cryopreservation method, namely scraping several cyclosporins from a fresh slant culture medium, transferring the scraped cyclosporins into a glycerol tube filled with 1.5mL of 30% sterilized glycerol, and performing cryopreservation at the temperature of-70 ℃.

Or freeze-preserving skimmed milk, which is to scrape several annuluses from fresh slant culture medium, transfer into glycerol tube filled with sterilized skimmed milk, and freeze-preserving at-70 deg.C.

The Tessaracococcus oleiagri DH10 strain screened by the invention has been preserved in China Center for Type Culture Collection (CCTCC) at 04/19/2021, and the addresses are as follows: the preservation number of Wuhan university in Wuhan City of China is: CCTCC NO: m2021404.

Example 2 decomposition of montmorillonite by DH10 Strain

1. Bacterial strain

The obtained Tessaracococcus oleiagri DH10 strain was selected in example 1.

2. Culture medium

The composition of the culture medium is: 10-50g/L of sucrose, 10-40g/L of sodium acetate, 3-20g/L of sodium lactate, 40.1-2g/L of MgSO40, 2-18g/L of KCl, 9-9 g/L of MnSO 45, 45-10 g/L of CuSO, 45-12 g/L of ZnSO, 41-7 g/L of KH2PO, 2-10g/L of montmorillonite and pH of 5.0-9.0. Sterilizing at 0.1MPa for 30min after the preparation of the culture medium.

3. Fermentation culture

The slant preserved strain is streaked and activated by inoculating loop on a flat plate, cultured for 20h at the constant temperature of 35 ℃, then three-loop strain (each inoculating loop strain contains more than 3 single colonies with obvious characteristics) is picked from the flat plate and inoculated into a shake flask (50mL triangular flask, the liquid loading is 30mL), cultured for 8h at 35 ℃ and 180r/min, the strain is centrifugally collected, is washed by sterile water for 3 times and then resuspended, inoculated into a montmorillonite culture medium, and anaerobically cultured at 35 ℃,

4. reduction of Fe (III) in clay mineral by microorganism

The Fe (II) content testing method adopts a phenanthroline spectrophotometry, and the specific operation refers to the measurement of HJ/T345-2007 ironAnd (2) determining phenanthroline spectrophotometry, after the Tessaracoccus oleiagri DH10 strain is inoculated, determining the total Fe content in the sample according to a standard method, taking 5mL of extract at intervals after culture, calculating the Fe (II) content by using a ferrous content determination standard curve, and calculating the Fe (III) reduction efficiency in the sample. After inoculation of the Tessaracococcus oleiagri DH10 strain, Fe in montmorillonite was compared to that in the blank control group²⁺The change in concentration with time is shown in FIG. 2. The result shows that the Tessaracococcus oleiagri DH10 can realize the high-efficiency reduction of Fe (III) in the montmorillonite, and the reduction efficiency is 41.9%.

5. Microscopic morphology observation of clay mineral before and after microbial action

And observing the structure, the morphology and the mineralogical changes of the clay mineral before and after the action of the Tessaracococcus oleiagri DH10 by using a scanning electron microscope.

The sample preparation method is as follows: (1) taking 1mL of logarithmic phase bacterial liquid in a 1.5mL EP tube, carrying out centrifugation at 13400r/min for 5min at 4 ℃, and discarding the supernatant; (2) washing with 1mL PBS with pH of 7.4, 13400r/min, 5min, centrifuging at 4 ℃, discarding supernatant, and repeating for 3 times; (3) placing the precipitate on a clean glass slide, and fixing the sample for 1h by using 2% paraformaldehyde-2.5% glutaraldehyde as a fixing solution; (4) dehydrating the sample with ethanol with different concentrations, wherein the ethanol concentration is 25%, 50%, 75%, 95% and 100%, and the ethanol concentration is used for twice, and the dehydration time is 15-20 min; (5) after the dehydration treatment is finished, putting the mixture into a vacuum freeze dryer for treatment for 24 hours; (6) spraying gold on the dried sample in vacuum; (7) and observing by using a scanning electron microscope to obtain the micro-morphology of the montmorillonite before and after the action of the microorganism under different magnifications.

The observation result of the scanning electron microscope is shown in FIG. 3, wherein FIG. 3-A is the original montmorillonite sample, and FIG. 3-B is the montmorillonite sample after 7 days of action of the strain DH 10. The results show that the original montmorillonite sample is relatively compact solid particles which are gathered together, and the montmorillonite sample is in a loose net structure after the Tessaracoccus oleiagri DH10 strain acts for 7 days, namely the montmorillonite sample has obvious corrosion phenomenon after the Tessaracoccus oleiagri DH10 strain acts.

Example 3 measurement of anti-swelling ratio

On the basis of example 2, the swelling properties of the smectite minerals before and after the action of the strain Tessaracococcus oleiagri DH10, Fe (III) reducing bacteria, were measured with reference to the method for measuring the clay swelling prevention rate in a standard clay stabilizer. The experimental steps are as follows: firstly, weighing 0.1g of dried control group montmorillonite sample, filling the sample into a 2.0mL centrifuge tube, and recording the volume V0 of the sample before expansion in the centrifuge tube; then adding 1.5mL of expansion medium solution (distilled water and kerosene are respectively used as expansion medium solution) and fully shaking up; standing at normal temperature for 24h, placing into a centrifuge, centrifuging at 5000 rpm for 15 min, and reading out the volume V1 of the sample after expansion. The above procedure was repeated to measure the swelling volume of the montmorillonite samples of the experimental group, and the volume before and after swelling was recorded as V2 and V3, respectively. The formula for calculating the swelling inhibition rate of the montmorillonite sample is as follows:

in formula (1): eta is the anti-swelling rate,%; v0 is the volume, mL, of the control dried montmorillonite sample; v1 is the swelling volume, mL, of control montmorillonite in swelling medium; v2 is the volume of dried montmorillonite after action of microorganism, mL; v3 is the swelling volume, mL, of microbially acted montmorillonite in swelling medium.

Experimental results show that the Tessaracococcus oleiagri DH10 strain has a good effect of inhibiting hydration swelling of clay minerals, wherein the swelling inhibition rate in a water system is 50.61%, and the swelling inhibition rate in a kerosene system is 87.63%. Namely, the Tessaracoccus oleiagri DH10 strain has a good effect of inhibiting clay swelling.

Example 4 iron-reducing bacteria strain DH10 reduction of reservoir injection pressure

Firstly, testing the permeability of a low-permeability reservoir by using formation water, then injecting a strain DH10 cell suspension, and culturing for 2 weeks; and then testing the permeability of the low-permeability reservoir by using formation water, thereby evaluating the influence of the iron dissimilatory bacteria DH10 on the injection pressure of the low-permeability reservoir. The specific operation steps are as follows:

1. baking and weighing the core, then placing the core into a core holder, and carrying out annular pressing and vacuumizing on the core holder;

2. injecting formation water at the flow rate of 0.50mL/min and the injection volume of 15PV, and recording the pressure difference between the inlet and the outlet of the core holder in the stable period;

3. injecting strain DH10 cell suspension at flow rate of 0.30mL/min, injecting volume of 2PV, closing inlet and outlet, and culturing at 55 deg.C for 2 weeks;

4. then, the formation water is injected with the flow rate of 0.50mL/min and the injection volume of 15PV, and the pressure difference between the inlet and the outlet of the core holder in the stable period is recorded.

The core parameters of the strain DH10 for evaluation of reduction of injection pressure of low permeability reservoirs are shown in Table 2, and the measurement results are shown in Table 3.

TABLE 2 evaluation of core parameters by iron reducing bacteria DH10 reducing reservoir injection pressure

TABLE 3 evaluation results of iron-reducing bacteria DH10 for lowering reservoir injection pressure

The results show that for the core mD2 with a permeability <10mD, the pressure difference at displacement with formation water is 0.305 MPa; after the iron reducing bacteria DH10 are used for acting, the pressure difference is reduced to 0.239MPa, and the injection pressure difference is reduced by 21.64%, which shows that the iron reducing bacteria DH10 can obviously reduce the injection pressure of a low-permeability reservoir.

Example 5 enhanced oil recovery by iron-reducing bacteria strain DH10

A rock core simulation experiment is used for evaluating the effect of the DH10 strain on improving the crude oil recovery efficiency, and the concrete steps are as follows:

1. connecting the core pretreatment and displacement devices;

2. saturated formation water with flow rate of 0.50mL/min and injection volume of 15 PV;

3. saturated crude oil with the flow rate of 0.50mL/min is observed, an outlet is observed until no formation water is produced, the original oil content of a rock core is calculated according to the volume of effluent water, and the rock core is aged for 72 hours at the temperature of 55 ℃;

4. and (3) carrying out primary water drive on formation water at the flow rate of 0.50mL/min until the water content of the outlet of the rock core is 98%, and calculating the volume of produced oil, namely the volume of the produced oil in primary water drive.

5. The control group was injected with formation water supplemented with 15mM sodium acetate, and the experimental group was injected with DH10 cell suspension; injecting 2PV at a flow rate of 0.30mL/min, closing the inlet and the outlet, and culturing at 55 deg.C for 2 weeks;

6. and then, displacing by using stratum water, wherein the flow rate is 0.50mL/min, and performing water displacement until the water content of the outlet of the rock core is 100%, so as to calculate the volume of produced oil, which is the volume of the produced oil of the secondary water displacement.

Core parameters for enhanced oil recovery evaluation by iron-reducing bacteria DH10 are shown in Table 4, and evaluation results are shown in Table 5.

TABLE 4 evaluation of core parameters for enhanced oil recovery by iron reducing bacteria DH10

TABLE 5 evaluation results of enhanced oil recovery by iron-reducing bacteria DH10

The results show that the recovery ratio of the primary water-drive rock cores ER0, ER1 and ER2 is 48.57%, 46.38% and 44.44% respectively by using the rock cores with the permeability of 70-100 mD; after the treatment by using the iron reducing bacteria enriched material DH10, the recovery ratio of secondary water flooding is respectively 0.71%, 6.52% and 5.56%; compared with the control group ER0, the recovery ratio of the experimental group ER1 and the recovery ratio of the ER2 are respectively improved by 5.81 percent and 4.84 percent, and the average value is 5.32 percent.

In conclusion, the iron reducing bacterium Tessaracococcus oleiagri DH10 is obtained by separation and screening, and can effectively decompose and corrode montmorillonite minerals and inhibit clay swelling; meanwhile, core experiments show that the Tessaracoccus oleiagri DH10 can reduce the water injection pressure in oil development, and the method is applied to crude oil recovery, so that the recovery rate of crude oil is obviously improved.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.