Sphingomonas paucimobilis sldt21 and application thereof

Abstract

The present application relates to a strain of Sphingomonas stellata SLDT21 and its application. The strain of Sphingomonas stellata SLDT21 has a preservation number of CGMCC No.17709. The Sphingomonas stellata SLDT21 is applied to oil production, microbial oil production and as a biological polysaccharide oil displacement agent. The present application has the following beneficial effects: (1) the strain of Sphingomonas stellata SLDT21 can grow and metabolize to produce a biological polysaccharide oil displacement agent under reservoir conditions, effectively plug large pores of the reservoir, realize liquid flow diversion and improve the on-site oil production effect; (2) the Sphingomonas stellata SLDT21 is applied to microbial single-well stimulation, and the average single-well oil increment is greater than 300 tons, the effective period is more than 200 days, and it is suitable for stimulation of high-water-cut single wells with high difficulty; (3) the Sphingomonas stellata SLDT21 is applied to microbial oil displacement, and the average daily oil increment of a well group is more than 10 tons, and the water cut reduction reaches 5%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of energy biotechnology and environmental biotechnology, and particularly relates to a strain of Sphingomonas paucimobilis SLDT21 and application thereof. BACKGROUND

[0002] With the continuous deepening of oilfield development, the water content of oil wells increases, the heterogeneity of oil reservoirs intensifies, and large channels are formed in the oil reservoirs, causing ineffective circulation of injected water.

[0003] At present, for high water cut oil reservoirs, the existing methods mainly realize the plugging of heterogeneous oil layers and large channels through mechanical layering and chemical plugging. However, the number of oil layers that can meet mechanical layering is limited, and chemical plugging mainly uses polymer gel, but polymer gel is greatly affected by reservoir temperature and salinity, and polymer plugging is mainly concentrated in the near-wellbore zone of water wells, and cannot achieve effective plugging in the deep part of the reservoir, and the effective period is limited. Therefore, a more effective technology is needed to achieve deep plugging of the reservoir, improve the percolation channel of crude oil, and improve the development effect of the block.

[0004] By screening special polysaccharide-producing functional strains from the reservoir, using the growth and metabolism of polysaccharide-producing functional strains in the reservoir, the percolation channel is changed, the liquid flow is diverted in the subsequent water flooding process, and the swept area of remaining oil is expanded. The reported microbial genera that can produce extracellular polysaccharides include Sphingomonas, Xanthomonas, and Schizophyllan. For example, Chinese Patent Application CN107058187A discloses a Sphingomonas paucimobilis strain and its application method, which provides a new Sphingomonas paucimobilis strain FJAT-10625, and the yield of extracellular polysaccharide obtained by using the strain is high, thereby increasing the source of extracellular polysaccharide-producing strains. Chinese Patent CN103421718B discloses a Sphingomonas paucimobilis strain and its application method, which is an endophyte isolated and screened from the wild micro-pore grass seeds in the Qinghai-Tibet Plateau, and can be used for industrial production of gellan gum, providing a new way for industrial production of endophyte gellan gum.

[0005] The above patents all disclose the screening of Sphingomonas paucimobilis and its application, but there are still the following deficiencies:

[0006] (1) The existing patent only discloses the screening and production method of Sphingomonas paucimobilis, but lacks specific and detailed application conditions and examples;

[0007] (2) the present disclosure does not involve the application of Sphingomonas paucimobilis in oil fields, and it is necessary to develop new fields in the field application of oil fields;

[0008] (3) the existing public disclosure only involves general functional bacteria, and does not involve specific preserved bacteria and application conditions. SUMMARY

[0009] The first object of the present application is to provide a Sphingomonas paucimobilis SLDT21.

[0010] Technical scheme: a Sphingomonas paucimobilis SLDT21, the preservation number of which is CGMCC No.17709.

[0011] A microbial agent, the active ingredient of which is the above-mentioned Sphingomonas paucimobilis.

[0012] Application of substance I and / or substance II and / or substance III and / or substance IV and / or substance V in oil production;

[0013] Substance I is the above-mentioned Sphingomonas paucimobilis;

[0014] Substance II is the above-mentioned microbial agent;

[0015] Substance III is the bacterial suspension of the above-mentioned Sphingomonas paucimobilis;

[0016] Substance IV is the culture solution of the above-mentioned Sphingomonas paucimobilis;

[0017] Substance V is the fermentation product of the above-mentioned Sphingomonas paucimobilis.

[0018] Application of substance I and / or substance II and / or substance III and / or substance IV and / or substance V in microbial oil production;

[0019] Substance I is the above-mentioned Sphingomonas paucimobilis;

[0020] Substance II is the above-mentioned microbial agent;

[0021] Substance III is the bacterial suspension of the above-mentioned Sphingomonas paucimobilis;

[0022] Substance IV is the culture solution of the above-mentioned Sphingomonas paucimobilis;

[0023] Substance V is the fermentation product of the above-mentioned Sphingomonas paucimobilis.

[0024] Application of Substance I and / or Substance II and / or Substance III and / or Substance IV and / or Substance V as biopolysaccharide flooding agents in microbial enhanced oil recovery;

[0025] Substance I is the aforementioned Sphingosine monocytogenes;

[0026] Substance II is the aforementioned bacterial agent;

[0027] Substance III is a bacterial suspension of the aforementioned Sphingomonas pyrenoidosa;

[0028] Substance IV is the culture medium of the aforementioned Sphingomonas pyogenes;

[0029] Substance V is the fermentation product of the aforementioned Sphingosine monocytogenes.

[0030] Furthermore, the *Sphingosine monocytogenes* strain produces the aforementioned biopolysaccharide oil-displacing agent in a nutrient culture medium containing glucose as a carbon source, yeast extract and sodium nitrate as nitrogen sources, dipotassium hydrogen phosphate as a phosphorus source, and magnesium sulfate and potassium chloride.

[0031] Furthermore, in the application, the nutrient culture medium is: glucose 40-60 g / L, yeast extract 1-3 g / L, sodium nitrate 1-3 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.2-0.7 g / L, potassium chloride 0.2-0.7 g / L, and pH value 6.0-7.5;

[0032] The preferred concentrations are: glucose 50 g / L, yeast extract 2.0 g / L, sodium nitrate 2.0 g / L, dipotassium hydrogen phosphate 1.5 g / L, magnesium sulfate 0.4 g / L, potassium chloride 0.5 g / L, and pH 6.0.

[0033] Furthermore, in the aforementioned application, the fermentation conditions of *Sphingosine Monoclonalella* are as follows: inoculum size 5%-15%, inoculum age 10-15 h, initial pH 6.0-7.5, temperature 30℃-60℃, stirring speed 200-300 rpm, and aeration rate 0.1-0.5 m³ / h. 3 / h, fermentation time 40-60h;

[0034] The preferred fermentation conditions for the *Sphingosine Monoclonalella* strain are: inoculum size 10%, inoculum age 12 h, initial pH 7.0, temperature 40 °C, stirring speed 240 rpm, and aeration rate 0.2 m³ / h. 3 / h, fermentation time is 50h.

[0035] Furthermore, in the aforementioned applications, the microbial enhanced oil recovery includes microbial single-well huff and puff or microbial flooding.

[0036] Furthermore, in the aforementioned application, the specific steps of the single-well microbial huff and puff process are as follows:

[0037] Use 50-100 ml of the prepared 5-20% Sphingosine monocytogenes fermentation broth from the above-mentioned strain. 3 and nutrient culture medium 50-200m 3 Injected into the formation from the oil well at a rate of 5–20 m / s. 3 / h, after the well is shut in for cultivation for 10-30 days, it is put into production. The bio-polysaccharide oil displacement agent produced by the reproduction and metabolism of Sphingosine Monoclonalella SLDT21 is used to improve the production capacity of a single well.

[0038] Furthermore, in the aforementioned application, the concentration of the *Sphingosine Monoclonalella* fermentation broth is 10–15%.

[0039] Furthermore, in the aforementioned application, the microbial single-well injection is a bio-polysaccharide oil displacement agent of *Sphingosine Monoclonalella* injected into the formation through an oil well, wherein:

[0040] The oil well is a high water-cut oil well with a water cut of 90-98%.

[0041] Furthermore, in the aforementioned application, the single-well microbial throughput includes the following steps:

[0042] Apply 0.1-0.5% of the biopolysaccharide oil-displacing agent from *Sphingomonas sphingosine monocytogenes* at a concentration of 150-300 mg / L. 3 The oil is injected into the formation from the well, and after a shut-in reaction period of 10–30 days, the well is opened for production. The bio-polysaccharide solid oil displacement agent is used to seal the large water inlet channels.

[0043] Furthermore, in the aforementioned application, the microbial single-well huff and puff involves injecting the aforementioned *Sphingomonas sphingosine monocytogenes* fermentation broth, nutrient culture medium, and biosurfactant into the formation through an oil well, synergistically enhancing single-well productivity.

[0044] The oil well is a high water-cut oil well with a water cut of 85-98%.

[0045] Furthermore, in the aforementioned application, the single-well microbial throughput includes the following steps:

[0046] Add 50-100 ml of the above-mentioned Sphingosine monocytogenes fermentation broth at a concentration of 5-20%. 3 and nutrient culture medium 50-200m 3 After injecting the oil into the formation and shutting in the well for 3-7 days, inject another 100-300m³ of biosurfactant fermentation fluid. 3 Injected into the formation from the oil well at a rate of 5–20 m / s. 3 / h, after the well is shut in for cultivation for 10-30 days, it is put into production. The biopolysaccharide oil displacement agent produced by the growth and metabolism of Sphingosine monocytogenes is used to plug the large water inlet channels. The single well production capacity is synergistically improved by the stripping and washing effect of biosurfactants.

[0047] Furthermore, in the aforementioned applications, the biosurfactant is one of lipopeptides, rhamnolipids, and sophorolipids;

[0048] The concentration of the *Sphingosine Monoclonalis* fermentation broth is 10–15%.

[0049] Furthermore, in the aforementioned application, the formula for calculating the injection volume of *Sphingosine Monoclonalella* fermentation broth is as follows:

[0050] V = 3.14R 2 Hфβ, where:

[0051] The injection volume of V-Sphingosine monocytogenes fermentation broth, in m³ 3 ;

[0052] R—processing radius, in meters;

[0053] H—Effective thickness, in meters (m);

[0054] ф—porosity;

[0055] β—usage coefficient, 1.0-1.2 for vertical and inclined wells, and 0.3-0.6 for horizontal wells.

[0056] Furthermore, in the aforementioned application, the microbial flooding involves injecting the fermentation broth of *Sphingomonas oligosporus* from a water injection well into the oil reservoir. The growth and metabolism of *Sphingomonas oligosporus* effectively seals large pores, increasing the sweep range of subsequent water flooding. Specifically:

[0057] The microbial injection rate is 50–120 m. 3 / d.

[0058] Furthermore, in the aforementioned application, the formula for calculating the injection volume of *Sphingosine Monoclonalella* fermentation broth is as follows:

[0059] V = 3.14L 2 Hфβ, where:

[0060] The injection volume of V-Sphingosine monocytogenes fermentation broth, in m³ 3 ;

[0061] L—Sweep distance, in meters, and is 0.2-0.5 times the distance between oil and water wells;

[0062] H—Effective thickness, in meters (m);

[0063] ф—porosity;

[0064] β—Dosage coefficient, with a value of 0.3-0.5.

[0065] Furthermore, in the aforementioned application, the microbial flooding involves injecting *Sphingomonas sphingosine monocytogenes* fermentation broth, nutrient culture medium, and biosurfactants into the oil reservoir from a water injection well. The growth and metabolism of *Sphingomonas sphingosine monocytogenes* effectively seals large pores, increasing the sweep range of subsequent water flooding. Furthermore, the stripping and washing effect of the biosurfactants synergistically enhances the productivity of the well group.

[0066] The injection rate of microbial flooding is 50–120 m. 3 / d;

[0067] The nutrient culture medium is as follows: glucose 40-60 g / L, yeast extract 1-3 g / L, sodium nitrate 1-3 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.2-0.7 g / L, potassium chloride 0.2-0.7 g / L, and pH value 6.0-7.5.

[0068] The preferred composition is 50 g / L glucose, 2.0 g / L yeast extract, 2.0 g / L sodium nitrate, 1.5 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 0.5 g / L potassium chloride, and pH 6.0.

[0069] The biosurfactant is one of lipopeptides, rhamnolipids, and sophorolipids.

[0070] Furthermore, in the aforementioned application, the calculation formulas for the injection amounts of *Sphingosine Monoclonalella* fermentation broth, nutrient culture medium, and biosurfactant are as follows:

[0071] V = 3.14L 2 Hфβ, where:

[0072] The sum of the injection volumes of *Sphingosine Monoclonalella vulgaris* fermentation broth, nutrient medium, and biosurfactant, in units of m³. 3 ,in:

[0073] The volume ratios of Sphingosine Monoclonalella fermentation broth, nutrient culture medium, and biosurfactant were (0.5–1): (0.5–1): (0.5–1);

[0074] L—Sweep distance, in meters, and is 0.2-0.5 times the distance between oil and water wells;

[0075] H—Effective thickness, in meters (m);

[0076] ф—porosity;

[0077] β—Dosage coefficient, with a value of 0.3-0.5.

[0078] A biopolysaccharide oil displacement agent, the active ingredients of which are substance I and / or substance II and / or substance III and / or substance IV and / or substance V;

[0079] Substance I is the aforementioned Sphingosine monocytogenes;

[0080] Substance II is the aforementioned bacterial agent;

[0081] Substance III is a bacterial suspension of the aforementioned Sphingomonas pyrenoidosa;

[0082] Substance IV is the culture medium of the aforementioned Sphingomonas pyogenes;

[0083] Substance V is the fermentation product of the aforementioned Sphingosine monocytogenes;

[0084] The function of the biopolysaccharide oil displacement agent is as follows (a), (b), or (c);

[0085] (a) Oil extraction;

[0086] (b) Microbial enhanced oil recovery;

[0087] (c) Biopolysaccharide oil displacement agent.

[0088] A method for preparing a bio-polysaccharide oil displacement agent includes the following steps:

[0089] (1) The above-mentioned *Sphingosine monocytogenes* was fermented in a nutrient medium containing glucose as the carbon source, yeast extract and sodium nitrate as the nitrogen source, dipotassium hydrogen phosphate as the phosphorus source, and magnesium sulfate and potassium chloride to obtain a fermentation broth, wherein:

[0090] The inoculum size for *Sphingosine monocytogenes* is 5%-15%, the inoculation period is 10-15 hours, the initial pH is 6.0-7.5, the temperature is 30℃-60℃, the stirring speed is 200-300 rpm, and the aeration rate is 0.1-0.5 m³ / h. 3 / h, fermentation time 40-60h;

[0091] (2) Add 90-95 v / v% ethanol to the fermentation broth obtained in step (1), stir thoroughly until fiber precipitate appears, and let stand for 1-2 hours.

[0092] (3) The fiber precipitate was separated by centrifugation to obtain the crude product of biopolysaccharide oil displacement agent;

[0093] (4) Wash the crude product of biopolysaccharide oil displacement agent with alcohol again, and dry the precipitate to constant weight to obtain biopolysaccharide oil displacement agent.

[0094] The advantages and beneficial effects of this invention are as follows:

[0095] (1) The present invention provides a strain of Sphingosine Monoclonalella SLDT21 that can grow and metabolize under reservoir conditions to produce biopolysaccharide oil displacement agent, which can effectively block the large channels of the reservoir, realize the liquid flow diversion, and improve the on-site oil production effect.

[0096] (2) The Sphingomonas SLDT21 was applied to microbial single-well huff and puff, with an average single-well oil increase of more than 300 tons and an effective period of more than 200 days. It is also suitable for huff and puff of high water-cut single wells with high difficulty.

[0097] (3) When the Sphingomonas SLDT21 was applied to microbial enhanced oil recovery, the average daily oil production of the well group increased by more than 10 tons, and the water cut decreased by 5%. Attached Figure Description

[0098] Figure 1 This is a microscopic morphological image of a strain of Sphingosine monocytogenes disclosed in this invention.

[0099] Figure 2 This is a colony morphology diagram of a strain of Sphingosine monocytogenes disclosed in this invention.

[0100] Figure 3 This is an evolutionary clade diagram of a *Sphingosine Monoclonalella* strain disclosed in this invention.

[0101] Figure 4 This is a diagram showing the state of the fermentation broth of a strain of Sphingosine Monoclonalella disclosed in this invention.

[0102] Preservation of biological materials for patent procedures:

[0103] Date of preservation: May 5, 2019

[0104] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0105] Address of the depositary: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences

[0106] Collection Number: CGMCC No. 17709

[0107] Classification and nomenclature: Sphingomonas paucimobilis. Detailed Implementation

[0108] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. With regard to numerical ranges, the endpoint values ​​of each range, and the endpoint values ​​of each range with individual point values, can be gradually combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this application.

[0109] The nutrient culture medium used in the following specific examples is as follows: glucose 40-60 g / L, yeast extract 1-3 g / L, sodium nitrate 1-3 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.2-0.7 g / L, potassium chloride 0.2-0.7 g / L, and pH value 6.0-7.5.

[0110] The preferred nutrient culture medium is: 50 g / L glucose, 2.0 g / L yeast extract, 2.0 g / L sodium nitrate, 1.5 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 0.5 g / L potassium chloride, and pH 6.0.

[0111] The specific embodiments of the present invention are described in detail below.

[0112] Example 1

[0113] Acquisition and Identification of Sphingosine Monoclonalella SLDT21 of the Present Invention

[0114] I. Obtaining Sphingosine Monoclonalella SLDT21

[0115] The Sphingomonas SLDT21 of this invention was isolated from the produced fluid of an oil well in a certain block of the Shengli Oilfield.

[0116] II. Identification of Sphingosine Monoclonalella SLDT21

[0117] The experimental methods were followed according to Bergey's Mannual of Systematic Bacteriology. The size and morphology of the cells, the presence or absence of flagella and spores, the growth temperature, and the biochemical characteristics such as catalase, starch hydrolysis, gelatin liquefaction, galactose, xylose, and lactose were observed.

[0118] The research results indicate that, as Figure 1 , Figure 2 As shown, the strain has long rod-shaped cells, no peritrichous flagella, is motile, and has round colonies that are white or slightly yellow. The cells are long rod-shaped and measure (0.2–0.4) × (2–10) μm.

[0119] Physiological and biochemical characteristics of Sphingosine monocytogenes SLDT21: aerobic and facultative anaerobic, growth temperature 30-60℃, optimal growth temperature 50℃, growth pH range 5-10, optimal growth pH range 7-7.5, and NaCl tolerance 0-15%.

[0120] Based on Bergey's Mannual of Systematic Bacteriology, a phylogenetic tree was constructed according to its morphological and physiological-biochemical characteristics, as well as the alignment results of the 16S rDNA gene sequence of this bacterium in GenBnk. Figure 3 Analysis revealed that SLDT21 is a new bacterium belonging to Sphingomonaspaucimobilis, with the Latin name Sphingomonaspaucimobilis.

[0121] Example 2

[0122] Performance evaluation parameters of Sphingosine Monoclonalella SLDT21:

[0123] The ability of in-situ growth and polysaccharide production under reservoir conditions, the ability of fermentation broth to improve the resistance coefficient and residual resistance coefficient of seepage, and the value of improved recovery through physical simulation experiments.

[0124] Performance evaluation method of Sphingosine monocytogenes SLDT21:

[0125] (1) Evaluation of growth and polysaccharide production capacity under reservoir conditions

[0126] Sphingosine monocytogenes SLDT21 was inoculated into nutrient medium at a 10% inoculum and cultured at 40°C for 48 hours. Bacterial density and viscosity of the fermentation broth were then tested. A schematic diagram of the fermentation broth is shown below. Figure 4 As shown in the table below, the specific experimental data are as follows:

[0127] Table 1. Culture of Sphingosine Monoclonalella (SMC)

[0128] Incubation time Bacterial density (cells / mL) Viscosity (mPa.s) 12h 1.6 x 10 7 ]]> 25 24h 2.4 x 10 8 ]]> 136 36h 6.6 x 10 8 ]]> 2650 48h 9.8 x 10 8 ]]> 5600

[0129] (2) Evaluation of the enhanced oil recovery value from physical simulation experiments of fermentation broth

[0130] ① Core preparation: Filling and sterilizing the core, and measuring air permeability;

[0131] ② Vacuum pumping and saturation simulation of formation water were performed to determine the core PV (pore volume);

[0132] ③ Saturated crude oil, core aged for 7 days, calculate the bound water saturation;

[0133] ④ For a single water drive, the water drive is carried out until the produced water has a water content of more than 98%, and the recovery rate of the single water drive is calculated.

[0134] ⑤ Inject 0.2-0.5 PV of Sphingosine Monoclonalella SLDT21 fermentation broth, and the calculated resistance coefficient is 17.5;

[0135] ⑥ Secondary water flooding, water flooding until the product liquid contains 100% water, the calculated residual resistance coefficient is 6.1, and the calculated improvement in displacement efficiency is 12.3%.

[0136] Performance of Sphingosine Monoclonalella SLDT21: Under reservoir temperature conditions, bacterial concentration greater than 10 8 The volume of the fermentation broth was 17.5 / mL, and the viscosity after fermentation reached 5600 mPa·s. The resistance coefficient of the fermentation broth to improve seepage was 17.5. The residual resistance coefficient remained at 6.1. The physical simulation experiment improved the displacement efficiency by 12.3%.

[0137] Example 3

[0138] Application of Sphingomyelin-Lystrophomonas SLDT21 as a biopolysaccharide flooding agent in microbial enhanced oil recovery.

[0139] In the above applications, the *Sphingosine monocytogenes* strain is fermented in a nutrient medium containing glucose as a carbon source, yeast extract and sodium nitrate as nitrogen sources, dipotassium hydrogen phosphate as a phosphorus source, and magnesium sulfate and potassium chloride.

[0140] The nutrient culture medium consisted of 40 g / L glucose, 1.0 g / L yeast extract, 1.0 g / L sodium nitrate, 0.5 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 0.5 g / L potassium chloride, and a pH of 6.0.

[0141] In another embodiment, the nutrient culture medium consists of 50 g / L glucose, 2.0 g / L yeast extract, 2.0 g / L sodium nitrate, 1.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.2 g / L potassium chloride, and a pH of 7.0.

[0142] In the third embodiment, the nutrient culture medium consists of 60 g / L glucose, 3.0 g / L yeast extract, 3.0 g / L sodium nitrate, 2.0 g / L dipotassium hydrogen phosphate, 0.7 g / L magnesium sulfate, 0.7 g / L potassium chloride, and a pH of 7.5.

[0143] In this embodiment, the fermentation conditions for *Sphingosine Monoclonalella* were: inoculum size 10%, inoculum age 10 h, initial pH 7.0, temperature 40°C, stirring speed 240 rpm, and aeration rate 0.1 m³ / h. 3 / h, fermentation time is 50h.

[0144] In another embodiment, the fermentation conditions for the *Sphingosine Monoclonalella* were: inoculum size 5%, inoculum age 15 h, initial pH 7.5, temperature 50°C, stirring speed 200 rpm, and aeration rate 0.5 m³ / h. 3 / h, fermentation time is 40h.

[0145] In the third embodiment, the fermentation conditions for the *Sphingosine Monoclonalella* were: inoculum size 15%, inoculum age 12 h, initial pH 6.0, temperature 60°C, stirring speed 300 rpm, and aeration rate 0.3 m³ / h. 3 / h, fermentation time is 60h.

[0146] A biopolysaccharide oil displacement agent, the active ingredients of which are substance I and / or substance II and / or substance III and / or substance IV and / or substance V;

[0147] Substance I is the aforementioned Sphingosine monocytogenes;

[0148] Substance II is the aforementioned bacterial agent;

[0149] Substance III is a bacterial suspension of the aforementioned Sphingomonas pyrenoidosa;

[0150] Substance IV is the culture medium of the aforementioned Sphingomonas pyogenes;

[0151] Substance V is the fermentation product of the aforementioned Sphingosine monocytogenes;

[0152] The function of the biopolysaccharide oil displacement agent is as follows (a), (b), or (c);

[0153] (a) Oil extraction;

[0154] (b) Microbial enhanced oil recovery;

[0155] (c) Biopolysaccharide oil displacement agent.

[0156] Example 4

[0157] A method for preparing a biopolysaccharide solid oil displacement agent includes the following steps:

[0158] (1) The *Sphingosine monocytogenes* strain described above was fermented in a nutrient medium containing glucose as the carbon source, yeast extract and sodium nitrate as the nitrogen source, dipotassium hydrogen phosphate as the phosphorus source, and magnesium sulfate and potassium chloride.

[0159] In this embodiment, the inoculum size of *Sphingosine monocytogenes* was 5%, the inoculation age was 15 hours, the initial pH was 7.0, the temperature was 40°C, the stirring speed was 250 rpm, and the aeration rate was 0.5 m³ / h. 3 / h, fermentation time 40h;

[0160] In another embodiment, the inoculum size of *Sphingosine monocytogenes* was 10%, the inoculation period was 10 hours, the initial pH was 6.0, the temperature was 50°C, the stirring speed was 200 rpm, and the aeration rate was 0.1 m³ / s. 3 / h, fermentation time 60h;

[0161] In the third embodiment, the inoculum size of *Sphingosine monocytogenes* was 15%, the inoculation period was 12 hours, the initial pH was 7.5, the temperature was 60°C, the stirring speed was 300 rpm, and the aeration rate was 0.3 m³ / h. 3 / h, fermentation time is 50h.

[0162] (2) Add 95% ethanol to the fermentation liquid, stir thoroughly until the fibers are visible to the naked eye, and let stand for 1-2 hours.

[0163] (3) Centrifuge to separate the precipitate and obtain the crude product of biological polysaccharide solid oil displacement agent;

[0164] (4) The crude product of the biopolysaccharide solid oil displacement agent is washed with alcohol once more, and the precipitate is dried at 60°C to constant weight to obtain the biopolysaccharide solid oil displacement agent.

[0165] A biopolysaccharide oil displacement agent is obtained by fermentation culture of the aforementioned Sphingomonas sphingosine monocytogenes. It is a solid biopolysaccharide product extracted from the liquid fermentation broth, which is beneficial for on-site transportation and construction.

[0166] The nutrient culture medium is composed of: glucose 40 g / L, yeast extract 3 g / L, sodium nitrate 2 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.2 g / L, potassium chloride 0.5 g / L, and pH 7.0.

[0167] In another embodiment, the nutrient culture medium is: 60 g / L glucose, 1 g / L yeast extract, 3 g / L sodium nitrate, 0.5 g / L dipotassium hydrogen phosphate, 0.7 g / L magnesium sulfate, 0.2 g / L potassium chloride, and pH 6.5.

[0168] In the third embodiment, the nutrient culture medium is: glucose 50 g / L, yeast extract 2 g / L, sodium nitrate 1 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate 0.5 g / L, potassium chloride 0.7 g / L, and pH value 7.5.

[0169] Example 5

[0170] The application of the fermentation broth of Sphingosine Monoclonalella SLDT21 provided by this invention in well A1 of a certain block in Shengli Oilfield.

[0171] Oil well overview: Formation temperature 65℃, formation water salinity 18800 mg / L, effective oil layer thickness 4.8 m, porosity 34%, permeability 850 × 10⁻⁶.-3 μm 2 The underground crude oil viscosity is 255 mPa·s, with a daily liquid output of 15 cubic meters and a daily oil output of 1.2 tons. The water content is 92%.

[0172] The above application refers to microbial single-well huff and puff.

[0173] Furthermore, the microbial single-well huff and puff involves injecting fermentation broth of Sphingomonas sphingosine monocytogenes SLDT21 and nutrient culture medium into the formation through an oil well, thereby utilizing the fermentation broth of Sphingomonas sphingosine monocytogenes SLDT21 to increase single-well productivity through reproduction and metabolism.

[0174] Furthermore, the specific steps of the aforementioned microbial single-well throughput are as follows:

[0175] Use 100ml of the prepared 10% Sphingosine monocytogenes SLDT21 fermentation broth. 3 and 100ml of nutrient culture medium 3 Injected into the formation from the oil well at a rate of 10m. 3 / h, after the well is shut in and cultured for 10 days, it is put into production. The bio-polysaccharide oil displacement agent produced by the reproduction and metabolism of Sphingosine Monoclonalella SLDT21 is used to improve the production capacity of a single well.

[0176] The nutrient culture medium consisted of 40 g / L glucose, 1.0 g / L yeast extract, 1.0 g / L sodium nitrate, 0.5 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 0.5 g / L potassium chloride, and a pH of 6.0.

[0177] In another embodiment, the nutrient culture medium consists of 50 g / L glucose, 2.0 g / L yeast extract, 2.0 g / L sodium nitrate, 1.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.2 g / L potassium chloride, and a pH of 7.0.

[0178] In the third embodiment, the nutrient culture medium consists of 60 g / L glucose, 3.0 g / L yeast extract, 3.0 g / L sodium nitrate, 2.0 g / L dipotassium hydrogen phosphate, 0.7 g / L magnesium sulfate, 0.7 g / L potassium chloride, and a pH of 7.5.

[0179] The specific implementation steps are as follows: Calculate the injection volume of SLDT21 fermentation broth and nutrient culture medium based on the effective thickness, porosity, and treatment radius of the oil layer in the oil well.

[0180] Total injection amount formula: V = 3.14R 2 Hфβ, where V—injection volume, m 3 R—treatment radius, 6m; H—effective thickness, m; ф—porosity (taken as 0.34); β—dosage coefficient (taken as 1.1), 200m 3The prepared SLDT21 fermentation broth and nutrient culture medium were injected into the formation from the oil well using a pump truck. The oil well was shut in for cultivation for 10 days before being opened for production.

[0181] Test results: The well's fluid and oil production both increased, with daily fluid production increasing from 15 cubic meters to 20.4 cubic meters and daily oil production increasing from 1.2 tons to 3.5 tons. The water cut decreased from 92% to 82.8%, a decrease of 9.2 percentage points. The cumulative oil production increased by 1,650 tons, and the effective period reached more than 200 days. The field test results were good.

[0182] Example 6

[0183] The application of the biopolysaccharide solid oil displacement agent of Sphingomonas SLDT21 provided by this invention in well B1 of a certain block of Shengli Oilfield.

[0184] Well Overview: Formation temperature 55℃, formation water salinity 9500 mg / L, effective oil layer thickness 6.2 m, porosity 36%, permeability 1650 × 10⁻⁶. -3 μm 2 The underground crude oil viscosity is 95 mPa·s, with a daily liquid volume of 23 cubic meters and a daily oil volume of 0.8 tons, containing 96.5% water.

[0185] The above application refers to microbial single-well huff and puff.

[0186] Furthermore, the microbial single-well huff and puff involves injecting a biopolysaccharide solid oil displacement agent of Sphingomonas sphingosine monocytogenes SLDT21 into the formation through an oil well. The high viscosity generated after the biopolysaccharide solid oil displacement agent dissolves is used to block and adjust high-permeability channels, thereby increasing the single-well productivity.

[0187] Furthermore, the specific steps of the aforementioned microbial single-well throughput are as follows:

[0188] Use 300ml of the prepared 0.3% concentration of Sphingomonas sphingosine monocytogenes SLDT21 biopolysaccharide solid oil displacement agent. 3 Injected into the formation from the oil well at a rate of 10m. 3 / h, the well is shut in for 10 days of cultivation and then put into production. The biopolysaccharide solid oil displacement agent of Sphingomonas SLDT21 is used to improve the production capacity of a single well.

[0189] The specific implementation steps are as follows: Calculate the injection volume of SLDT21 fermentation broth and nutrient culture medium based on the effective thickness, porosity, and treatment radius of the oil layer in the oil well.

[0190] Total injection amount formula: V = 3.14R 2 Hфβ, where V—injection volume, m 3R—treatment radius, 6.2m; H—effective thickness, m; ф—porosity (taken as 0.36); β—dosage coefficient (taken as 1.1), 300m 3 The prepared bio-polysaccharide solid oil displacement agent is injected into the formation from the oil well using a pump truck. After the oil well is shut in and incubated for 10 days, it is then put back into production.

[0191] Test results: The well's fluid and oil production both increased. Daily fluid production decreased from 23 cubic meters to 16 cubic meters, daily oil production increased from 0.8 tons to 1.5 tons, and water cut decreased from 96.5% to 90.6%, a decrease of 5.9 percentage points. The cumulative oil production increased by 850 tons, and the effective period reached more than 300 days. The field test results were good.

[0192] Example 7

[0193] The application of the fermentation broth of Sphingosine Monoclonalella SLDT21 provided by this invention in well C1 of a certain block of Shengli Oilfield.

[0194] Oil well overview: Formation temperature 38℃, formation water salinity 52540mg / L, effective oil layer thickness 7m, porosity 28%, permeability 2584×10 -3 μm 2 The underground crude oil viscosity is 2246 mPa·s, with a daily liquid volume of 32 cubic meters and a daily oil volume of 1.3 tons, containing 95.9% water.

[0195] The above application refers to microbial single-well huff and puff.

[0196] Furthermore, the microbial single-well huff and puff involves injecting Sphingomonas sphingosine monocytogenes fermentation broth, nutrient culture medium, and biosurfactants into the formation through an oil well, thereby synergistically enhancing the single-well productivity.

[0197] Furthermore, the aforementioned microbial single-well throughput includes the following steps:

[0198] 50 ml of 15% Sphingomonas sphingosine monocytogenes SLDT21 fermentation broth was added. 3 and 100ml of nutrient culture medium 3 After injecting the oil into the formation and shutting in the well for 5 days, inject another 200 mg of biosurfactant. 3 Injected into the formation from the oil well at a rate of 20m. 3 / h, after the well is shut in for 20 days of cultivation, it is put back into production. The biopolysaccharide oil displacement agent produced by the growth and metabolism of Sphingomonas sphingosine monocytogenes SLDT21 is used to plug the large water inlet channels. The single well production capacity is synergistically improved by the stripping and washing effect of biosurfactants.

[0199] Further, the biosurfactant is sophorolipid. In another embodiment, the biosurfactant is a lipopeptide. In yet another embodiment, the biosurfactant is rhamnolipid.

[0200] The specific implementation steps are as follows: Calculate the injection volume of SLDT21 fermentation broth, nutrient culture medium and biosurfactant based on the effective thickness, porosity and treatment radius of the oil layer in the oil well.

[0201] Total injection amount formula: V = 3.14R 2 Hфβ, where V—injection volume, m 3 R—treatment radius, 7.2m; H—effective thickness, m; ф—porosity (taken as 0.36); β—dosage coefficient (taken as 1.1), 350m 3 The prepared bio-polysaccharide solid oil displacement agent is injected into the formation from the oil well using a pump truck. The oil well is then shut in and incubated for 15 days before being put back into production.

[0202] Test results: The well's fluid and oil production both increased. Daily fluid production decreased from 32 cubic meters to 21 cubic meters, daily oil production increased from 1.3 tons to 3.5 tons, and water cut decreased from 95.9% to 83.3%, a decrease of 12.6 percentage points. The cumulative increase in oil production was 2080 tons, and the effective period exceeded 200 days. The field test results were good.

[0203] Example 8

[0204] The fermentation broth of Sphingosine Monoclonalella SLDT21 provided by this invention has been applied in Block D of Shengli Oilfield.

[0205] Oil well overview: Formation temperature 50℃, formation water salinity 12500mg / L, effective oil layer thickness 5.4m, porosity 34%, permeability 430×10⁻⁶. -3 μm 2 The underground crude oil viscosity is 145 mPa.s. The well group consists of 1 injection and 3 production wells. The distance between oil and water wells is 240 m. The daily production of liquid in the block is 34 cubic meters, the daily production of oil is 3.6 t, and the overall water cut is 89.4%.

[0206] In this field application, it refers to microbial drive.

[0207] Furthermore, the microbial flooding involves injecting the fermentation broth of Sphingomonas sphingosine monocytogenes SLDT21 from the injection well. The growth and metabolism of Sphingomonas sphingosine monocytogenes SLDT21 effectively seals large pores, increasing the sweep range of subsequent water flooding and thus improving the production capacity of different oil wells in the well group.

[0208] Furthermore, the formula for calculating the injection volume of Sphingomonas sphingosine monocytogenes SLDT21 fermentation broth is as follows: V = 3.14 L 2 Hфβ=3.14×120×5.4×0.34×0.3=207.5m 3

[0209] Furthermore, the injection speed is 50m.3 / d, the specific steps include:

[0210] A 10% concentration of Sphingomonas sphingosine monocytogenes SLDT21 fermentation broth was injected into a 207.5m³ well. 3 After injection, stop injection for 7 days, then resume normal water injection.

[0211] Test results: After the test, the production of fluid and oil in this well group increased. The fluid production increased by 3 cubic meters / day, the production increased by 2.5 tons / day, the water cut decreased by 5.9 percentage points, and the effective period reached more than 300 days. The field test results were good.

[0212] Table 2. Well group effects after on-site implementation

[0213]

[0214] Example 9

[0215] The fermentation broth, nutrient culture medium, and biosurfactant of Sphingosine Monoclonalella SLDT21 provided by this invention are applied in Block E of Shengli Oilfield.

[0216] Oil well overview: Formation temperature 80℃, formation water salinity 42500mg / L, effective oil layer thickness 8.9m, porosity 38%, permeability 1690×10⁻⁶. -3 μm 2 The underground crude oil viscosity is 275 mPa·s. The well group consists of 1 injection and 4 production wells. The distance between oil and water wells is 210 m. The daily production of liquid in the block is 65 cubic meters, the daily production of oil is 3.6 t, and the overall water cut is 94.5%.

[0217] In this field application, it refers to microbial drive.

[0218] Furthermore, the microbial flooding involves injecting Sphingomonas sphingosine monocytogenes SLDT21 fermentation broth, nutrient culture medium, and biosurfactants from the injection well. The growth and metabolism of Sphingomonas sphingosine monocytogenes SLDT21 effectively seals large pores, increasing the sweep range of subsequent water flooding. Furthermore, the stripping and washing effect of the biosurfactants synergistically improves the production capacity of the well group.

[0219] Furthermore, the calculation formula for the injection volume of Sphingomonas sphingosine monocytogenes SLDT21 fermentation broth, nutrient medium, and biosurfactant is as follows: V = 3.14 L 2 Hфβ=3.14×84×8.9×0.38×0.5=446m 3

[0220] Furthermore, the injection speed is 100m. 3 / d, the specific steps include:

[0221] 100 ml of 15% Sphingosine monocytogenes SLDT21 fermentation broth was added. 3 200ml of nutrient culture medium 3 and biosurfactants 146m 3 (That is, the volume ratio of the three is 0.5:1:0.73), totaling 446m³. 3 Inject 4 cubic meters of water from the injection well, stop injection for 15 days after completion, and then resume normal water injection.

[0222] Test results: After the test, the production of fluid and oil in this well group increased. The fluid volume increased by 10 cubic meters / day, the production increased by 4 tons / day, the water cut decreased by 4.6 percentage points, and the effective period reached more than 240 days. The field test results were good.

[0223] Table 3. Well group effects after on-site implementation

[0224]

[0225] 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 inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Application of Substance I and / or Substance II and / or Substance III and / or Substance IV as biopolysaccharide flooding agents in microbial enhanced oil recovery; Substance I is Sphingomonas paucimobilis SLDT21, and the preservation number of Sphingomonas paucimobilis SLDT21 is CGMCC No.17709; Substance II is a bacterial agent, and the active ingredient of the bacterial agent is the *Sphingomonas sphingosine monocytogenes* SLDT21. Substance III is a bacterial suspension of the aforementioned Sphingomonas SLDT21; Substance IV is the culture medium of *Sphingomonas sphingosine monocytogenes* SLDT21, wherein: The *Sphingomonas sphingosine monocytogenes* SLDT21 is produced as a biopolysaccharide oil-displacing agent in a nutrient medium containing glucose as a carbon source, yeast extract and sodium nitrate as nitrogen sources, dipotassium hydrogen phosphate as a phosphorus source, and magnesium sulfate and potassium chloride. Microbial oil recovery includes microbial single-well huff and puff or microbial flooding.

2. In the application described in claim 1, the characteristic is that, The nutrient culture medium is as follows: glucose 40-60 g / L, yeast extract 1-3 g / L, sodium nitrate 1-3 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.2-0.7 g / L, potassium chloride 0.2-0.7 g / L, and pH value 6.0-7.

5.

3. In the application described in claim 2, the characteristic is that, The nutrient culture medium is: The concentrations are as follows: glucose 50 g / L, yeast extract 2.0 g / L, sodium nitrate 2.0 g / L, dipotassium hydrogen phosphate 1.5 g / L, magnesium sulfate 0.4 g / L, potassium chloride 0.5 g / L, and pH 6.

0.

4. In the application described in claim 1, the characteristic is that, The fermentation conditions for *Sphingosine Monoclonalella* SLDT21 were as follows: inoculum size 5%-15%, inoculum age 10-15 h, initial pH 6.0-7.5, temperature 30℃-60℃, stirring speed 200-300 rpm, and aeration rate 0.1-0.5 m³ / h. 3 / h, fermentation time is 40-60h.

5. In the application described in claim 4, the characteristic is that, The fermentation conditions for *Sphingosine Monoclonalella SLDT21* were as follows: inoculum size 10%, inoculum age 12 h, initial pH 7.0, temperature 40℃, stirring speed 240 rpm, and aeration rate 0.2 m³ / h. 3 / h, fermentation time is 50h.

6. The application as described in claim 1, characterized in that, The specific steps of the microbial single-well huff and puff process are as follows: Use 50-100 ml of the prepared 5-20% Sphingosine monocytogenes SLDT21 fermentation broth. 3 and nutrient culture medium 50-200m 3 Injected into the formation from the oil well at a rate of 5–20 m / s. 3 / h, after the well is shut in for cultivation for 10-30 days, it is put into production. The bio-polysaccharide oil displacement agent produced by the reproduction and metabolism of Sphingosine Monoclonalella SLDT21 is used to improve the production capacity of a single well.

7. The application as described in claim 6, characterized in that, The concentration of the fermentation broth of *Sphingomonas sphingosine monocytogenes* SLDT21 is 10–15%.

8. The application as described in claim 1, characterized in that, The microbial single-well injection is a bio-polysaccharide oil displacement agent of *Sphingomonas sphingosine monocytogenes* SLDT21 injected into the formation through an oil well, wherein: The oil well is a high water-cut oil well with a water cut of 90-98%.

9. The application as described in claim 8, characterized in that, The aforementioned microbial single-well throughput includes the following steps: Apply 0.1-0.5% of the biopolysaccharide oil-displacing agent from Sphingomonas SLDT21 at a concentration of 150-300 mg / L. 3 The oil is injected into the formation from the well, and after a shut-in reaction period of 10–30 days, the well is opened for production. The bio-polysaccharide solid oil displacement agent is used to seal the large water inlet channels.

10. The application as described in claim 1, characterized in that, The microbial single-well huff and puff involves injecting the fermentation broth of *Sphingosine Monoclonalella* SLDT21, nutrient culture medium, and biosurfactants into the formation through an oil well to synergistically enhance single-well productivity. Specifically: The oil well is a high water-cut oil well with a water cut of 85-98%.

11. The application as described in claim 10, characterized in that, The aforementioned microbial single-well throughput includes the following steps: Add 50-100 ml of the fermentation broth of *Sphingomonas sphingosine monocytogenes* SLDT21 at a concentration of 5-20%. 3 and nutrient culture medium 50-200m 3 After injecting the oil into the formation and shutting in the well for 3-7 days, inject another 100-300m³ of biosurfactant fermentation fluid. 3 Injected into the formation from the oil well at a rate of 5–20 m / s. 3 / h, after the well is shut in for cultivation for 10-30 days, it is put into production. The biopolysaccharide oil displacement agent produced by the growth and metabolism of Sphingomonas sphingosine monocytogenes SLDT21 is used to block the large water inlet channels. The single well production capacity is synergistically improved by the stripping and washing effect of biosurfactants.

12. The application as described in claim 11, characterized in that, The biosurfactant is one of lipopeptides, rhamnolipids, and sophorolipids; The concentration of the fermentation broth of *Sphingomonas sphingosine monocytogenes* SLDT21 is 10–15%.

13. The application as described in claim 11, characterized in that, The formula for calculating the injection volume of Sphingosine Monoclonalella SLDT21 fermentation broth is as follows: V = 3.14R 2 Hфβ, where: The injection volume of V-Sphingomonas SLDT21 fermentation broth, in m³ 3 ; R—processing radius, in meters; H—Effective thickness, in meters (m); ф—porosity; β—usage coefficient, 1.0-1.2 for vertical and inclined wells, and 0.3-0.6 for horizontal wells.

14. The application as described in claim 1, characterized in that, The microbial flooding involves injecting the fermentation broth of *Sphingomonas oligosporus* SLDT21 from a water injection well into the oil reservoir. The growth and metabolism of *Sphingomonas oligosporus* SLDT21 effectively seals large pores, increasing the sweep range of subsequent water flooding. Specifically: The microbial injection rate is 50–120 m. 3 / d.

15. The application as described in claim 1, characterized in that, The formula for calculating the injection volume of Sphingosine Monoclonalella SLDT21 fermentation broth is as follows: V = 3.14L 2 Hфβ, where: The injection volume of V-Sphingomonas SLDT21 fermentation broth, in m³ 3 ; L—Sweep distance, in meters, and is 0.2-0.5 times the distance between oil and water wells; H—Effective thickness, in meters (m); ф—porosity; β—Dosage coefficient, with a value of 0.3-0.

5.

16. The application as described in claim 1, characterized in that, The microbial flooding involves injecting Sphingomonas sphingosine monocytogenes SLDT21 fermentation broth, nutrient culture medium, and biosurfactants into the oil reservoir from a water injection well. The growth and metabolism of Sphingomonas sphingosine monocytogenes SLDT21 effectively seals large pores, increasing the sweep range of subsequent water flooding. Furthermore, the stripping and washing effect of the biosurfactants synergistically enhances the productivity of the well group. The injection rate of microbial flooding is 50–120 m. 3 / d; The nutrient culture medium is as follows: glucose 40-60 g / L, yeast extract 1-3 g / L, sodium nitrate 1-3 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, magnesium sulfate 0.2-0.7 g / L, potassium chloride 0.2-0.7 g / L, and pH value 6.0-7.

5. The biosurfactant is one of lipopeptides, rhamnolipids, and sophorolipids.

17. The application as described in claim 16, characterized in that, The nutrient culture medium consisted of: 50 g / L glucose, 2.0 g / L yeast extract, 2.0 g / L sodium nitrate, 1.5 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 0.5 g / L potassium chloride, and a pH of 6.

0.

18. The application as described in claim 16, characterized in that, The formulas for calculating the injection amounts of Sphingosine Monoclonalella SLDT21 fermentation broth, nutrient medium, and biosurfactant are as follows: V = 3.14L 2 Hфβ, where: The sum of the injection volumes of V-Sphingomonas SLDT21 fermentation broth, nutrient medium, and biosurfactant, in units of m. 3 ,in: The volume ratios of Sphingosine Monoclonalella SLDT21 fermentation broth, nutrient medium, and biosurfactant were (0.5–1): (0.5–1): (0.5–1); L—Sweep distance, in meters, and is 0.2-0.5 times the distance between oil and water wells; H—Effective thickness, in meters (m); ф—porosity; β—Dosage coefficient, with a value of 0.3-0.5.

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