Pseudomonas stutzeri strain dn-1 and its use

Abstract

The present application relates to a strain of pseudomonas stutzeri DN-1 and application thereof.A strain of pseudomonas stutzeri DN-1, the preservation number of which is CGMCC No.22969.The present application also discloses application of the pseudomonas stutzeri as an aid in oilfield oily sewage treatment, application in oil well produced liquid treatment, and application in oil well produced liquid treatment.The strain of pseudomonas stutzeri DN-1 provided by the present application can grow in situ under oil reservoir conditions, can effectively inhibit the breeding of SRB, reduce the corrosion of oil well, and has the advantages of convenient use, good effect, high economic benefit and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of environmental microbiology, and particularly relates to a Pseudomonas stutzeri DN-1 and application thereof. BACKGROUND

[0002] At present, most of the oilfields in China adopt water injection oil recovery process, with the development into the middle and late period, the water content of produced liquid is continuously increased, and the microbial corrosion is more and more serious. The growth, metabolism and reproduction of microorganisms can cause serious corrosion of equipment, pipelines and other metal materials, and can block the pipeline, damage the oil layer, cause the injection pressure to rise, and reduce the oil production and oil and gas quality, which brings great harm to the oilfield production. The microorganisms causing the corrosion of metal materials are mainly bacteria participating in the sulfur and iron element cycle in nature, namely anaerobic sulfate-reducing bacteria (SRB) and aerobic iron bacteria and sulfur bacillus.

[0003] The physical sterilization method and the chemical sterilization method are mainly used for killing SRB in oilfields. The physical sterilization method has remarkable killing effect on free SRB, but it is difficult to kill SRB at a long distance or SRB in an attached state due to the limitation of the action distance. In the chemical sterilization method, the widely used sterilizing agents mainly include quaternary ammonium salt, aldehyde, heterocyclic compound and their compound, but the sterilizing agents are difficult to penetrate because SRB is often protected in polysaccharide gum produced by other microorganisms. For general oxidizing sterilizing agents, the sterilizing agents are difficult to have effective sterilization effect because microorganisms are in a reducing environment of hydrogen sulfide. The existence of the biofilm reduces the sterilization efficiency and even causes the sterilization to be invalid, so that drug-resistant bacteria are produced.

[0004] The biological method is a method for inhibiting the activity of SRB through biological competition, changes the traditional thinking mode of simply pursuing the quantity of killed SRB, and turns to the purpose of inhibiting the activity of SRB, which is the innovation of the concept and method for controlling sulfide hazards in the oilfield system, and the biological method is less used in the oilfield system in China. By adding suitable denitrifying bacteria into the oil layer, the denitrifying bacteria can utilize the main carbon source on which SRB survives, so as to rapidly grow and reproduce, affect the reduction of sulfate by SRB, and achieve the purpose of inhibiting SRB. SUMMARY

[0005] The application provides a Pseudomonas stutzeri DN-1 and application thereof aiming at the deficiencies of the prior art.

[0006] Technical scheme: A Pseudomonas stutzeri DN-1, the preservation number of which is CGMCC No. 22969.

[0007] A bacterial agent, the active ingredient of which is the Pseudomonas stutzeri mentioned above.

[0008] A bacterial agent, comprising the Pseudomonas stutzeri mentioned above and a nutrient medium.

[0009] Further, the nutrient medium is glucose 1-8 g / L, methanol 1.5-3.0 g / L, sodium acetate 0.5-2.5 g / L, sodium nitrate 0.6-2.0 g / L, potassium dihydrogen phosphate 1-3 g / L, yeast powder 0.2-0.8 g / L, sodium chloride 3-8 g / L, and pH value is 7-8.

[0010] Still further, the nutrient medium is glucose 2-5 g / L, methanol 2-2.5 g / L, sodium acetate 1.5-2.0 g / L, sodium nitrate 0.8-1.5 g / L, potassium dihydrogen phosphate 1.2-2.0 g / L, yeast powder 0.3-0.5 g / L, sodium chloride 3-5 g / L, and pH value is 7-8.

[0011] Application of the substance I and / or the substance II and / or the substance III in oilfield oily sewage treatment;

[0012] The substance I is the Pseudomonas stutzeri mentioned above;

[0013] The substance II is the bacterial agent mentioned above;

[0014] The substance III is the fermentation liquor of the Pseudomonas stutzeri mentioned above.

[0015] Application of the substance I and / or the substance II and / or the substance III in oil well produced fluid treatment;

[0016] The substance I is the Pseudomonas stutzeri mentioned above;

[0017] The substance II is the bacterial agent mentioned above;

[0018] The substance III is the fermentation liquor of the Pseudomonas stutzeri mentioned above.

[0019] Application of the substance I and / or the substance II and / or the substance III as SRB inhibitor in oil well produced fluid treatment;

[0020] The substance I is the Pseudomonas stutzeri mentioned above;

[0021] The substance II is the bacterial agent mentioned above;

[0022] The substance III is the fermentation liquor of the Pseudomonas stutzeri mentioned above.

[0023] A product, the active ingredient of which is the substance I and / or the substance II and / or the substance III;

[0024] The substance I is the Pseudomonas stutzeri mentioned above;

[0025] The substance II is the microbial agent as described above.

[0026] The substance III is the fermentation liquor of the Pseudomonas stutzeri as described above.

[0027] The function of the product is as follows (a) or (b) or (c).

[0028] (a) oilfield oily sewage treatment.

[0029] (b) oil well produced liquid treatment.

[0030] (c) SRB inhibitor.

[0031] A method for inhibiting sulfate-reducing bacteria in oil well produced liquid, by continuously adding the substance I and / or the substance II and / or the substance III into the oil well injection liquid.

[0032] The substance I is the Pseudomonas stutzeri as described above.

[0033] The substance II is the microbial agent as described above.

[0034] The substance III is the fermentation liquor of the Pseudomonas stutzeri as described above.

[0035] Advantages and beneficial effects: the advantages and beneficial effects of the present application are as follows:

[0036] 1. The Pseudomonas stutzeri DN-1 provided by the present application has a growth temperature of 30-60 DEG C, can grow in situ under oil reservoir conditions, the microbial agent can effectively inhibit the breeding of SRB, and reduce the corrosion of oil wells (caused by hydrogen sulfide produced by sulfate-reducing bacteria), and the corrosion rate is reduced from 0.094 mm / a to 0.025 mm / a after use.

[0037] 2. The microbial agent is convenient to use and has good effect, and has high economic benefit, and can avoid the problems of large dosage, high cost and poor safety and environmental protection performance caused by the use of traditional chemical bactericides. DETAILED DESCRIPTION

[0038] Figure 1 It is a morphological diagram of the Pseudomonas stutzeri DN-1 strain disclosed by the present application.

[0039] Figure 2 It is a phylogenetic tree of the Pseudomonas stutzeri DN-1 disclosed by the present application.

[0040] Figure 3 It is the change of corrosion rate of the Pseudomonas stutzeri DN-1 disclosed by the present application before and after application in two oil wells in Chunliang oil production plant of Shengli oilfield. DETAILED DESCRIPTION

[0041] The specific embodiments of the present application are described in detail below.

[0042] Example 1

[0043] Screening of Pseudomonas stutzeri DN-1 strain

[0044] The oil well produced liquid with serious SRB breeding in Chunliang oil production plant was taken by a disposable syringe, 1ml was inoculated into the culture medium (the culture medium composition: glucose 2g / L, methanol 2g / L, sodium acetate 2g / L, sodium nitrate 1g / L, potassium dihydrogen phosphate 2g / L, yeast powder 0.5g / L, sodium chloride 5g / L, pH value is 7), and 80% nitrogen was blown off during the preparation of the culture medium to ensure the anaerobic environment), and 50℃ constant temperature culture was carried out for 7d until single colonies appeared on the culture medium. The single colonies on the culture medium were picked and transferred into the same component anaerobic liquid culture medium for expansion culture, and 50℃ constant temperature culture was carried out for 7d. The culture solution was inoculated into the anaerobic solid culture medium again, and 50℃ constant temperature culture was carried out for 7d, and single colonies were separated.

[0045] The well-grown single colonies were respectively inoculated into the same component anaerobic liquid culture medium, 50℃ constant temperature culture was carried out for 3d, and then the strains with strong denitrification capacity were screened by detecting the decrease of nitrate content in the culture medium, and finally a strain with the best denitrification effect was obtained, which was named as strain DN-1.

[0046] Example 2

[0047] Identification and preservation of Pseudomonas stutzeri DN-1

[0048] I. Morphology and physiological and biochemical characteristics of Pseudomonas stutzeri DN-1

[0049] 1. Test strain

[0050] The strain DN-1 isolated in Example 1 of the present application.

[0051] 2. Experimental method

[0052] The experimental method of Bergey's Manual of Systematic Bacteriology was referred to, and the gram staining, size and morphology of the bacterial body, growth temperature, growth PH range and NaCl tolerance were detected. The methyl red, contact enzyme, V-P, glucose acid production, starch hydrolysis, pyocyanin, oxidase experiment, nitrate reduction, nitrite reduction experiment, and experiments using acetate, citrate, arabinose, galactose, xylose, muscle sugar, lactose, rhamnose and sucrose were carried out.

[0053] 3. Experimental results

[0054] The results show that:

[0055] 1. Morphological characteristics of strain DN-1:

[0056] (1) Colony characteristics: The colony is round, milky white, smooth and moist on the surface.

[0057] (2) Cellular morphological characteristics: The cells are rod-shaped, with a single polar flagellum, and the size is (0.2 - 0.4) × (4 - 20) μm, as Figure 1 described.

[0058] 2. Physiological and biochemical characteristics of strain DN-1: Facultative anaerobic, growth temperature 30 - 60 °C, optimal growth temperature 50 °C, growth pH range 5 - 10, optimal growth pH range 7 - 8, NaCl tolerance 0 - 15%.

[0059] 3. The catalase test is positive, and the methyl red, V-P, acid production from glucose, starch hydrolysis, pyocyanin, and oxidase tests are all negative. It can reduce nitrates and nitrites, and can utilize acetate, citrate, arabinose, galactose, lactose, rhamnose, and sucrose, but cannot utilize xylose and inositol.

[0060] Partial physiological and biochemical characteristics of strain DN-1 are shown in Table 1:

[0061] Table 1 Partial physiological and biochemical characteristics of strain DN-1

[0062]

[0063]

[0064] 4. Referring to the content of "Bergey’s Mannual of Systematic Bacteriology", according to its morphological characteristics, physiological and biochemical characteristics, and referring to the alignment results of the 16S rDNA gene sequence of this bacterium in GenBnk, a phylogenetic tree ( Figure 2 ) was constructed for analysis, and it was identified that strain DN-1 belongs to a new species of the genus Pseudomonas stutzeri, specifically Pseudomonas stutzeri.

[0065] II. Preservation of Pseudomonas stutzeri DN-1

[0066] A strain of Pseudomonas stutzeri DN-1 provided by the present invention, hereinafter referred to as DN-1, was deposited on July २८, २०२१ at the "China General Microbiological Culture Collection Center" (Institute of Microbiology, Chinese Academy of Sciences, No. ३, Yard १, Beichen West Road, Chaoyang District, Beijing), and its deposit number is "CGMCC No. २२९६९", and the taxonomic name in Chinese is "Pseudomonas stutzeri", and the Latin name is "Pseudomonas stutzeri".

[0067] Example 3

[0068] Inhibition ability experiment of Pseudomonas stutzeri DN-1 on sulfate-reducing bacteria

[0069] 1. Test strain

[0070] The Pseudomonas stutzeri DN-1 strain isolated in Example 1 of the present application.

[0071] 2. Experimental method

[0072] The test strain was picked up as a single colony and inoculated into 100 ml of anaerobic liquid medium, and cultured at 37 DEG C overnight to the logarithmic growth phase, and the medium composition was the same as in Example 1. Four portions of oil well produced fluid containing different amounts of sulfate-reducing bacteria (SRB) were taken and placed in 100 mL anaerobic bottles, and the number of SRB was determined by MPN method to be 25 / mL, 600 / mL, 2500 / mL and 6000 / mL, respectively. The cultured bacterial liquid was inoculated into the four anaerobic bottles at an inoculation amount of 2%, and placed in a 37 DEG C constant temperature incubator, and the number of SRB was detected after 24 h.

[0073] 3. Test results

[0074] The test results are shown in Table 2. Under the condition of 37 DEG C, the initial number of sulfate-reducing bacteria (25-6000) / mL, after inoculation of the bacterial liquid for 24 h, the number of sulfate-reducing bacteria was reduced to (2.5-250) / mL, and the bacteria had obvious inhibitory effect on sulfate-reducing bacteria.

[0075] Table 2 Comparison of the number of sulfate-reducing bacteria before and after inoculation of Pseudomonas stutzeri DN-1

[0076] Sample No. Before inoculation (cells / mL) After inoculation (cells / mL) 1 25 2.5 2 600 25 3 2500 60 4 6000 250

[0077] Example 4

[0078] Application of Pseudomonas stutzeri DN-1 in two oil wells A-21 and A-35 of a block in Chunliang Oil Production Plant of Shengli Oilfield

[0079] 1. Test strain

[0080] The Pseudomonas stutzeri DN-1 strain isolated in Example 1 of the present application.

[0081] 2. Preparation of bacterial liquid

[0082] Pseudomonas stutzeri DN-1 was picked up from a single colony and inoculated into 100 ml of anaerobic liquid medium, and incubated at 37°C overnight to the logarithmic growth phase to obtain a bacterial solution, wherein:

[0083] The anaerobic liquid medium comprises 1 g / L of glucose, 1.5 g / L of methanol, 0.5 g / L of sodium acetate, 0.6 g / L of sodium nitrate, 1 g / L of potassium dihydrogen phosphate, 0.2 g / L of yeast powder, and 3 g / L of sodium chloride, and has a pH value of 7.

[0084] 3. Oil well profile

[0085] The two oil wells (A-21 and A-35) of Chunliang Oil Production Plant in Shengli Oilfield have high SRB content and high corrosion rate, with an average corrosion rate of 0.094 mm / a. In the early stage, a bactericide, dodecyl dimethyl benzyl ammonium chloride, was added at a concentration of 150 mg / L. After treatment, the bacterial concentrations of CL-21 and CL-35 were 1000 cells / mL and 2500 cells / mL, respectively, the bactericidal rates were 60% and 58.4%, respectively, and the corrosion rates were 0.052 mm / a and 0.050 mm / a, respectively. The bactericidal and corrosion prevention effects were not satisfactory. Before the addition of Pseudomonas stutzeri DN-1, the produced liquid had the water quality shown in Table 3.

[0086] Table 3 Analysis results of produced liquid water quality of two oil wells

[0087]

[0088] 3. Implementation steps

[0089] The bacterial solution was continuously added by an oil well dosing machine, and the bacterial solution and the produced liquid were fully contacted and reacted. Pseudomonas stutzeri DN-1 was added into the dosing machine at a concentration of 150 mg / L, and the addition was continuous.

[0090] 4. Test results

[0091] The SRB and corrosion rate were detected at the wellhead every 5 days. The number of SRB was finally reduced from 2500 cells / mL and 6000 cells / mL to 60 cells / mL and cells / mL. The corrosion rate of the two oil wells was controlled below 0.025 mm / a, the corrosion prevention rate was reduced by more than 73%, the bactericidal rate was more than 95%, and the bactericidal and corrosion prevention effects were obvious.

[0092] Example 5

[0093] Application of Pseudomonas stutzeri DN-1 in two oil wells B-45 and B-52 in a block of Chunliang Oil Production Plant of Shengli Oilfield.

[0094] 1. Test strain

[0095] The Pseudomonas stutzeri DN-1 strain isolated in the embodiment 1 of the present application.

[0096] 2. Preparation of bacterial solution

[0097] The Pseudomonas stutzeri DN-1 is picked up from a single colony and inoculated into 100 ml of anaerobic liquid medium, and cultured at 37℃ overnight to the logarithmic growth phase to obtain a bacterial solution, wherein:

[0098] The anaerobic liquid medium is composed of 8 g / L of glucose, 3.0 g / L of methanol, 2.5 g / L of sodium acetate, 2.0 g / L of sodium nitrate, 3 g / L of potassium dihydrogen phosphate, 0.8 g / L of yeast powder, and 8 g / L of sodium chloride, and the pH value is 8.

[0099] 3. Oil well profile

[0100] The two oil wells (B-45 and B-52) in Chunliang Oil Production Plant of Shengli Oilfield have high content of SRB and high corrosion rate, and the average corrosion rate reaches 0.081 mm / a. In the early stage, the bactericide dodecyl dimethyl benzyl ammonium chloride is added at a concentration of 120 mg / L. After treatment, the bacterial concentrations of B-45 and B-52 are 500 and 1500 per ml respectively, the sterilization rates are 75% and 70% respectively, the corrosion rates are 0.050 mm / a and 0.047 mm / a respectively, and the sterilization and corrosion prevention effects are not satisfactory. Before adding the Pseudomonas stutzeri DN-1, the produced liquid water quality is shown in Table 4.

[0101] Table 4 Analysis results of produced liquid water quality of two oil wells

[0102]

[0103] 3. Implementation steps

[0104] The bacterial solution is continuously added by an oil well dosing machine, and the bacterial solution and the produced liquid are fully contacted and reacted. The Pseudomonas stutzeri DN-1 bacterial solution is added into the dosing machine at a concentration of 120 mg / L, and the adding mode is continuous adding.

[0105] 4. Test results

[0106] The SRB and corrosion rate were detected every 5 days at the wellhead, the number of SRB was reduced from 2000 / mL, 5000 / mL to 50 / mL, 100 / mL, the corrosion rates of the two oil wells B-45, B-52 were 0.020 mm / a and 0.018 mm / a respectively, the corrosion prevention rate was reduced by more than 70%, the sterilization rate was more than 97%, the sterilization and corrosion prevention effects were obvious.

[0107] Example 6

[0108] The example 5 was substantially the same, the only difference was that the anaerobic liquid medium was different.

[0109] The anaerobic liquid medium was glucose 2 g / L, methanol 2 g / L, sodium acetate 1.5 g / L, sodium nitrate 0.8 g / L, potassium dihydrogen phosphate 1.2 g / L, yeast powder 0.3 g / L, sodium chloride 3 g / L, and the pH value was 7.5.

[0110] Example 7

[0111] The example 5 was substantially the same, the only difference was that the anaerobic liquid medium was different.

[0112] The anaerobic liquid medium was glucose 5 g / L, methanol 2.5 g / L, sodium acetate 2.0 g / L, sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 2.0 g / L, yeast powder 0.5 g / L, sodium chloride 5 g / L, and the pH value was 8.

[0113] The above has made the detailed description to the embodiment of the present application. However, the present application is not limited to the above embodiment, within the knowledge range possessed by the ordinary skilled in the art, various changes can be made without departing from the purpose of the present application.

Claims

1. A strain of Pseudomonas stutzeri DN-1, characterized in that, Its preservation number is CGMCC No. 22969.

2. An inoculant characterized in that, The active ingredient of the bacterial agent is the Pseudomonas stutzeri in claim 1.

3. An inoculant characterized in that, The bacterial agent comprises the Pseudomonas stutzeri in claim 1 and a nutrient medium.

4. The bacterial agent of claim 3, wherein The nutrient medium is glucose 1-8 g / L, methanol 1.5-3.0 g / L, sodium acetate 0.5-2.5 g / L, sodium nitrate 0.6-2.0 g / L, potassium dihydrogen phosphate 1-3 g / L, yeast powder 0.2-0.8 g / L, sodium chloride 3-8 g / L, and pH value is 7-8.

5. The bacterial agent of claim 4, wherein The nutrient medium is glucose 2-5 g / L, methanol 2-2.5 g / L, sodium acetate 1.5-2.0 g / L, sodium nitrate 0.8-1.5 g / L, potassium dihydrogen phosphate 1.2-2.0 g / L, yeast powder 0.3-0.5 g / L, sodium chloride 3-5 g / L, and pH value is 7-8.

6. Application of substance I and / or substance II as SRB inhibitor in the treatment of oilfield oily sewage; Substance I is the Pseudomonas stutzeri in claim 1; Substance II is the bacterial agent in claim 2.

7. Application of substance I and / or substance II as SRB inhibitor in the treatment of oil well produced fluid; Substance I is the Pseudomonas stutzeri in claim 1; Substance II is the bacterial agent in claim 2.

8. A product, whose active ingredient is substance I and / or substance II; Substance I is the Pseudomonas stutzeri in claim 1; Substance II is the bacterial agent in claim 2. The function of the product is as follows (a) or (b); (a) as SRB inhibitor in the treatment of oilfield oily sewage; (b) as SRB inhibitor in the treatment of oil well produced fluid.

9. A method of inhibiting sulfate-reducing bacteria in oilfield production fluids, characterized by, Substance I and / or substance II are added into the oil well injection fluid by continuous feeding; Substance I is the Pseudomonas stutzeri in claim 1; Substance II is the bacterial agent in claim 2.

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