CN112225324A - Screening method of nitrate reducing bacteria for demulsification of strong-emulsification ASP flooding produced water - Google Patents
Screening method of nitrate reducing bacteria for demulsification of strong-emulsification ASP flooding produced water Download PDFInfo
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Abstract
The invention belongs to the technical field of industrial microorganisms, and relates to a method for screening nitrate reducing bacteria for demulsification of strong-emulsification ASP flooding produced water. A method for screening nitrate reducing bacteria for demulsification of strongly-emulsified ternary combination flooding produced water comprises the following steps: (1) carrying out enrichment culture on the inoculum, and separating and purifying the product; (2) screening alkane degrading bacteria from the separated and purified culture; (3) further screening alkane degrading bacteria with nitrate reducing property from the obtained alkane degrading bacteria; (4) and (4) predicting the demulsification effect of the bacteria screened in the step (3) on the ASP flooding produced water by using a multiple linear regression equation. The invention provides a high-efficiency screening method of a microbial agent with alkane degradation, nitrate reduction and biological demulsification functions; the screening method can provide an important guiding function for the biological treatment of the ternary complex flooding produced water of the strong emulsification of the oil field, and effectively promotes the progress of the treatment technology of the ternary complex flooding produced water of the oil field.
Description
Technical Field
The invention belongs to the technical field of industrial microorganisms, and relates to a biological demulsifying bacteria screening method for strong-emulsification ASP flooding produced water.
Background
At present, most oil fields in China enter the later stage of water injection exploitation. In order to further improve the extraction rate of crude oil, the enhanced oil recovery technology including the ASP flooding oil recovery technology is widely applied. Wherein, the ASP flooding oil recovery technology can improve the oil recovery rate by more than 20 percent. The ASP flooding technology is already applied industrially in a plurality of oil fields including Daqing oil fields in China, but the application of the ASP flooding oil extraction technology produces a large amount of produced water containing surfactant, polymer, colloidal particles, emulsified oil, sulfate reducing bacteria and the like, and has the characteristics of strong emulsification and easy biological acidification.
The conventional oil-water separation method for water flooding and polymer flooding is difficult to meet the oil-water separation requirement of the ternary combination flooding produced water. The currently developed technologies including electric flocculation, precipitation adsorption, membrane filtration, air flotation, chemical demulsifier and the like have the defects of complex operation flow, high secondary pollution risk, high economic cost and the like. How to realize economic, efficient and green removal of strongly emulsified suspended oil drops with small particle size is the key of the ternary combination flooding produced water treatment.
The biological demulsifying microbial inoculum is an oil-water separation method which is environment-friendly and has high demulsifying efficiency and is used for demulsifying an emulsion by utilizing an active substance which is generated by microbial metabolism and has an emulsion demulsifying function. However, no report is found on the screening method of biological demulsifying bacteria aiming at the strong emulsification ASP flooding produced water at present.
The microorganism usually needs to add a nitrogen source in the process of producing the demulsifier, and the same demulsifying bacteria can also utilize various types of nitrogen sources, including: peptone, ammonium chloride, potassium nitrate, ammonium nitrate, etc. However, there are few reports on biological demulsifying bacteria having high-efficiency nitrate reduction characteristics.
Nitrate-reducing bacteria can be generally classified into heterotrophic nitrate-reducing bacteria and autotrophic nitrate-reducing bacteria. The heterotrophic nitrate reducing bacteria take organic matters as electron donors and nitrate as electron acceptors, and the nitrate is subjected to nitrite, nitric oxide and nitrous oxide to finally generate nitrogen. The microorganism may perform several or all of these steps. The autotrophic nitrate reducing bacteria take sulfide and the like as electron donors, nitrate as electron acceptors, the sulfide can be converted into elemental sulfur or sulfate, and the nitrate finally generates nitrogen gas through nitrite, nitric oxide and nitrous oxide. Both of these nitrate-reducing bacteria have the potential to control the biological acidification of oil reservoirs or produced water. Wherein the heterotrophic nitrate-reducing bacteria inhibit the activity of the sulfate-reducing bacteria by electron donor competition or metabolism of the intermediate nitrite. The autotrophic nitrate-reducing bacteria can degrade sulfides in the produced water or oil reservoirs. Meanwhile, the autotrophic nitrate reducing bacteria can also accumulate nitrite in the process of reducing nitrate, and the nitrite is utilized to further inhibit the activity of the sulfate reducing bacteria. By virtue of these effects, nitrate-reducing bacteria can be used to provide enhanced control of the biological acidification of oil reservoirs or produced water.
The ternary complex flooding produced water contains a large amount of emulsified oil and sulfate reducing bacteria, and if the demulsifying bacteria with nitrate reducing capability is used for demulsifying the strongly emulsified ternary complex flooding produced water, the species abundance of the nitrate reducing bacteria in the produced water can be enhanced to a certain extent, so that the biological acidification control process depending on the nitrate reducing bacteria is promoted.
Disclosure of Invention
The invention aims to provide a high-efficiency screening method of nitrate reducing bacteria aiming at demulsification of strong-emulsification ASP flooding produced water, aiming at the defects and problems in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that: a nitrate reducing bacteria screening method for demulsification of strongly emulsified ternary combination flooding produced water is characterized by comprising the following 4 steps:
(1) carrying out enrichment culture on the inoculum, and separating and purifying the product;
(2) screening alkane degrading bacteria from the separated and purified culture;
(3) further screening alkane degrading bacteria with nitrate reducing property from the obtained alkane degrading bacteria;
(4) and predicting the demulsification effect of the alkane degrading bacteria with the nitrate reduction characteristic on the strong-emulsification ASP flooding produced water by using a multivariate linear equation.
In a preferred mode of the invention, the inoculum is taken from activated sludge or crude oil contaminated oil sludge around an ASP produced water well.
Further preferably, the enrichment culture method comprises the following steps: adding 5-10 g of inoculum into an inorganic nutrient salt culture medium, and culturing for 5d at 30-35 ℃ and 120-200 rpm; and then sucking 2-10mL of enrichment liquid, adding the enrichment liquid into a new nutrient salt solution, and repeating the culture step for 2-3 times.
Further preferably, the inorganic nutrient salt culture medium comprises the following components: KNO3 3g/L，KH2PO4 4g/L，K2HPO43g/L，MgSO4·7H2O 0.2g/L，CaCl2 0.02g/L，FeCl30.05g/L, 2mL/L of trace element solution, 4% (v/v) of liquid paraffin, and pH 7.0.
As a preferred mode of the present invention, the alkane degrading bacteria screening method comprises: inoculating a loop of the isolated and purified culture in a stoppered test tube containing an inorganic salt culture medium, and culturing for 1d in a constant temperature incubator at 30-35 ℃ and 120-200 rpm.
In a preferred embodiment of the present invention, the method for screening alkane degrading bacteria having a nitrate reducing property comprises: inoculating alkane degrading bacteria into an inorganic salt nutrient medium with known nitrate concentration, and culturing in a constant temperature incubator for 3-5 days; centrifuging a proper amount of culture solution, and detecting the content of nitrate and nitrite in the supernatant; the bacteria with reduced nitrate content and accumulated nitrite are alkane degrading bacteria with nitrate reducing property.
As a preferred mode of the invention, the method for predicting the demulsification effect of the strains screened in the step (3) on the ASP flooding produced water by using the multiple linear regression equation comprises the following steps:
(1) constructing a demulsification effect multiple linear regression equation prediction model of the demulsifying bacteria on the ASP flooding produced water:
T＝-115.011+40.044×OD600+4.136×OS-0.401×Zeta-0.719×NO3 --Nrem.ratio
wherein NO3 --Nrem.ratioThe nitrate removal rate (%); OD600The absorbance value of the bacterial liquid of the target strain after the target strain is cultured in the inorganic salt nutrient solution for 5 days; OS is the size (cm) of an oil discharge ring of a bacterial liquid after the target bacterial strain is cultured in an inorganic salt nutrient solution for 5d, and Zeta is the Zeta potential (mV/m) of a bacterial suspension of the target bacterial strain; t is the light transmittance (%) of the water phase after the ternary combination flooding produced water is demulsified;
(2) detecting OD of the bacterial liquid obtained in the step (3)600A value;
(3) demulsifying the ASP flooding produced water by using the strain obtained in the step (3), and recording the diameter of a formed blank oil ring, wherein the diameter is OS (cm);
(4) detecting the zeta potential of the bacterial liquid obtained in the step (3);
(5) calculating the nitrate removal rate NO of the bacterial liquid obtained in the step (3)3 --Nrem.ratio(％)；
(6) And (4) calculating the light transmittance of the demulsified water phase by using the multiple linear regression equation, and further predicting the demulsification effect of the strain obtained in the step (3) on the ASP flooding produced water.
The nitrate reducing bacteria screening method for demulsification of the strong-emulsification ASP flooding produced water has the following beneficial effects:
(1) provides a high-efficiency screening method of microbial agents with alkane degradation, nitrate reduction and biological demulsification functions;
(2) the screening method can provide an important guiding function for the biological treatment of the ternary complex flooding produced water of the strong emulsification of the oil field, and effectively promotes the progress of the treatment technology of the ternary complex flooding produced water of the oil field;
(3) and predicting the demulsification capability of the screened biological demulsification bacteria by using a multiple linear regression equation, and verifying that the prediction result is highly consistent with the actual measurement result, thereby proving that the method can accurately predict the demulsification capability of the biological demulsification bacteria.
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FIG. 1 is a graph of the average particle size of oil droplets in simulated strong emulsion ASP flooding produced water;
FIG. 2 is a relation between a predicted value of a multiple linear regression equation and actually measured water phase light transmittance after demulsification;
FIG. 3 shows the demulsification effect of the strongly emulsified ASP flooding produced water.
Detailed Description
In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
One embodiment provided by the invention is as follows: a nitrate reducing bacteria screening method aiming at demulsification of strong emulsification ASP flooding produced water comprises the following steps:
(1) inoculating 10g of fresh activated sludge or crude oil polluted oil sludge around a ternary composite flooding extraction well into an inorganic salt nutrient medium, and culturing for 5d at the temperature of 30-35 ℃ and the rotating speed of 120-200 rpm; then sucking 2-10mL of enrichment culture product, adding the enrichment culture product into a new inorganic salt nutrient medium, and repeating the culture step for 2-3 times.
Wherein, the inorganic salt nutrient medium comprises the following components: KNO3 3g/L，KH2PO4 4g/L，K2HPO4 3g/L，MgSO4·7H2O 0.2g/L，CaCl2 0.02g/L，FeCl30.05g/L, 2mL/L of trace element solution, 4% (v/v) of liquid paraffin, and pH 7.0.
The trace element solution comprises the following components: ZnSO4 1.5g/L，CuSO4 0.15g/L，H3BO3 1.5g/L，MnSO41.5g/L。
And continuously carrying out enrichment culture to obtain a target microorganism mixed flora, separating and purifying all potential target strains by adopting an enrichment culture and adopting a yeast extract peptone agar solid culture medium.
(2) And (3) inoculating each purified target strain growing on a yeast extract peptone agar solid culture medium by using 1 inoculating loop into a test tube with a plug containing 2mL of inorganic salt nutrient culture medium, and culturing for 1d in an incubator at constant temperature (30-35 ℃) and at the rotating speed of 120-200 rpm.
The microorganism with obvious growth or emulsification is alkane degrading bacteria.
(3) And (3) continuing culturing the test tube which has obvious growth and emulsification phenomena in the step (2) for 4d at the constant temperature (30-35 ℃) and the rotating speed of 120-200 rpm. After 1 part of culture solution is centrifuged, the contents of nitrate and nitrite in water are detected (the contents of nitrate and nitrite are measured by a national standard method), and strains with obvious nitrate removal and nitrite accumulation are reserved.
(4) Randomly selecting 10 microorganisms with nitrate reducing capability obtained in the step (3), respectively inoculating the microorganisms into conical flasks containing 100mL of inorganic salt nutrient medium, and culturing for 5d in a constant-temperature (30-35 ℃) incubator at the rotating speed of 120-200 rpm.
(5) Detecting OD of all bacterial liquid obtained in the step (4) on an ultraviolet-visible spectrophotometer600The value is obtained.
(6) And (3) adding 100mL of deionized water into 1 culture dish with the diameter of 15cm, then dropwise adding 50 mu L of crude oil, after a stable oil film is formed, dropwise adding 50 mu L of the bacterial liquid with the nitrate reducing capability obtained in the step (4) into the center of the oil film, and recording the diameter of the formed blank oil ring after the oil ring is stabilized (about 1 min). This step was repeated 3 times, and the mean value was calculated as OS (cm).
(7) 10mL of the bacterial suspension obtained in step (4) was collected and centrifuged at 13000rpm to obtain bacterial cells. After washing with 10mL of n-hexane for 2 times, the cells were centrifuged at 13000rpm, and the obtained cells were resuspended in 10mL of deionized water. The ZETA potential (mV/m) of the bacterial suspensions was then measured separately using the ZETA PALS, Bruken, USA.
(8) 2mL of the bacterial suspension obtained in the step (4) was collected and centrifuged at 13000rpm to obtain a supernatant. Respectively detecting the nitrate content of the supernatant, and calculating the removal rate NO of the nitrate3 --Nrem.ratio(％)。
(9) And (3) respectively adding 2mL of the bacterial liquid obtained in the step (4) into a graduated tube containing 8mL of simulated strong emulsification ASP flooding produced water, swirling for 1min at 3000rpm, placing in a 35 ℃ water bath kettle, standing for 24h, and detecting the light transmittance T (%) at a position 5cm below the liquid level of the test tube after demulsification.
(10) Constructing a multiple linear regression equation prediction model of the demulsification effect of the demulsifying bacteria on the ASP flooding produced water, wherein the specific method comprises the following steps:
and (5) fitting and evaluating the data obtained in the steps (5) to (8) by using a multiple linear regression function of the R language to obtain the following multiple linear equation.
T＝-115.011+40.044×OD600+4.136×OS-0.401×Zeta-0.719×NO3 --Nrem.ratio
Wherein NO3 --Nrem.ratioThe nitrate removal rate (%); OD600The absorbance value of the bacterial liquid of the target strain after the target strain is cultured in the inorganic salt nutrient solution for 5 days; OS is the size (cm) of an oil discharge ring of a bacterial liquid after the target bacterial strain is cultured in an inorganic salt nutrient solution for 5 d; zeta is the Zeta potential (mV/m) of the bacterial suspension of the target bacteria; and T is the light transmittance (%) of the water phase after the ternary combination flooding produced water is demulsified.
(11) From the measured results, the strains with the light transmittance T greater than 15% can be identified as: the efficient ternary combination flooding produced aquatic organism demulsifying bacteria with nitrate reducing capability. This value can be used as the prediction threshold for the above multiple linear regression equation, i.e.: if the T obtained by equation prediction is more than 15%, the strain is judged to be nitrate reducing bacteria for strong emulsification ASP flooding produced water biological demulsification.
In order to evaluate the accuracy of the demulsification effect of the biological demulsifying bacteria on the ASP flooding produced water predicted by the multiple linear regression equation in the embodiment, the invention designs and provides the following verification test.
1. Preparation of simulated strong-emulsification ASP flooding produced water
First, an aqueous mineral solution is prepared. Taking 1600mg/L NaCl and NaHCO according to the mass ratio3 2600mg/L，Na2CO3300mg/L，MgSO4 400mg/L，CaCl220mg/L, NaOH 400mg/L, HPAM (MW: 300 ten thousand) 400mg/L, petroleum sulfonate solution (provided by Shengli oil field, wherein the petroleum sulfonate content is about 50%) 200mL, and mild stirring for 1 h.
2. 1.5mL of crude oil was added to 500mL of an aqueous mineral solution by volume and emulsified in a shear emulsifier (Blender LB20EG, Waring Commercial, U.S. A.) at high speed (rotational speed grade: 7) for 5 min. The strong emulsification three-component combination flooding produced water with the average oil drop particle size of 65nm is prepared. Figure 1 shows the oil droplet size distribution of a simulated strong emulsion ASP flooding produced water.
3. Culture of demulsifying bacteria
(1) Preparing inorganic salt nutrient solution, which comprises the following components: KNO3 3g/L，KH2PO4 4g/L，K2HPO4 3g/L，MgSO4·7H2O 0.2g/L，CaCl2 0.02g/L，FeCl30.05g/L, 2mL of trace element solution, 4mL of liquid paraffin, 1000mL of deionized water and pH 7.0. Autoclaving at 121 deg.C for 15 min. And cooling for later use.
(2) Inoculating all target strains into a conical flask containing 100mL of inorganic salt nutrient solution, and culturing for 5d at the temperature of 30-35 ℃ and the rotating speed of 120-200 rpm.
(3) Respectively adding 2mL of microbial culture solution into 8 portions of graduated tubes containing simulated strong-emulsification ASP flooding produced water, and uniformly mixing at 3000rpm for 1 min. And (3) placing the test tube in a water bath kettle at 35 ℃, standing for 24 hours, and detecting the light transmittance of the test tube at a position 5cm below the liquid level after demulsification.
4. And comparing the measured result with the prediction result of the multiple linear regression equation. Fig. 2 shows the relationship between the predicted result and the measured result of the multiple linear regression equation, and it can be known from the figure that: the regression equation accurately predicts the demulsification effect of the obtained target strain on the strong emulsification ASP flooding produced water.
FIG. 3 shows the biological demulsification effect of the strong-emulsification ASP flooding produced water. As can be seen from the figure: the microbial agent (bacterium 1, bacterium 2, bacterium 3, bacterium 4, bacterium 6, bacterium 7 and bacterium 8) obtained by the method has excellent demulsification effect on the strongly emulsified ASP flooding produced water.
Claims (7)
1. A method for screening nitrate reducing bacteria for demulsification of strongly emulsified ternary combination flooding produced water is characterized by comprising the following steps:
(1) carrying out enrichment culture on the inoculum, and separating and purifying the product;
(2) screening alkane degrading bacteria from the separated and purified culture;
(3) further screening alkane degrading bacteria with nitrate reducing property from the obtained alkane degrading bacteria;
(4) and (4) predicting the demulsification effect of the bacteria screened in the step (3) on the ASP flooding produced water by using a multiple linear regression equation.
2. The screening method of nitrate reducing bacteria for demulsification of strongly-emulsified three-component combined drive produced water as claimed in claim 1, wherein the inoculum is taken from activated sludge or oil sludge polluted by crude oil around a three-component combined drive produced water well.
3. The screening method of nitrate reducing bacteria for demulsification of strongly-emulsified ternary combination flooding produced water according to claim 1, wherein the enrichment culture method comprises the following steps: adding 5-10 g of inoculum into 100mL of inorganic nutrient salt culture medium, and culturing for 5d at 30-35 ℃ and 120-200 rpm; and then sucking 2-10mL of enrichment liquid, adding the enrichment liquid into a new nutrient salt solution, and repeating the culture step for 2-3 times.
4. The screening method of nitrate reducing bacteria for demulsification of strongly-emulsified ternary combination flooding produced water according to claim 3, wherein the inorganic nutrient salt culture medium comprises the following components: KNO3 3g/L，KH2PO44g/L，K2HPO4 3g/L，MgSO4·7H2O 0.2g/L，CaCl2 0.02g/L，FeCl30.05g/L, 2mL/L of trace element solution, 4% (v/v) of liquid paraffin, and pH 7.0.
5. The screening method of nitrate reducing bacteria for demulsification of strongly-emulsified ternary combination flooding produced water according to claim 1, wherein the screening method of alkane degrading bacteria is as follows: inoculating a loop of separated and purified culture in a stoppered test tube containing an inorganic salt culture medium, and culturing for 1d in a constant-temperature incubator at 30-35 ℃ under the condition of 120-200 rpm.
6. The screening method of nitrate reducing bacteria for demulsification of strongly-emulsified ternary combination flooding produced water according to claim 1, wherein the screening method of alkane degrading bacteria with nitrate reducing property is as follows: inoculating alkane degrading bacteria into an inorganic salt nutrient medium with known nitrate concentration, and culturing in a constant temperature incubator for 3-5 days; centrifuging a proper amount of culture solution, and detecting the content of nitrate and nitrite in the supernatant; the bacteria with reduced nitrate content and accumulated nitrite are alkane degrading bacteria with nitrate reducing property.
7. The method for screening the nitrate reducing bacteria for demulsifying the strongly-emulsified ASP flooding produced water according to claim 1, wherein the step of predicting the demulsification effect of the target bacteria screened in the step (3) on the ASP flooding produced water by using a multiple linear regression equation comprises the following steps:
(1) constructing a demulsification effect multiple linear regression equation prediction model of the demulsifying bacteria on the ASP flooding produced water:
T＝-115.011+40.044×OD600+4.136×OS-0.401×Zeta-0.719×NO3 --Nrem.ratio
wherein NO3 --Nrem.ratioThe nitrate removal rate (%); OD600The absorbance value of the bacterial liquid of the target strain after the target strain is cultured in the inorganic salt nutrient solution for 5 days; OS is the size (cm) of an oil discharge ring of a bacterial liquid after the target bacterial strain is cultured in an inorganic salt nutrient solution for 5d, and Zeta is the Zeta potential (mV/m) of a bacterial suspension of the target bacterial strain; t is the light transmittance (%) of the water phase after the ternary combination flooding produced water is demulsified;
(2) detecting OD of the bacterial liquid obtained in the step (3)600A value;
(3) demulsifying the ASP flooding produced water by using the strain obtained in the step (3), and recording the diameter of a formed blank oil ring, wherein the diameter is OS (cm);
(4) detecting the zeta potential (mV/m) of the bacterial liquid obtained in the step (3);
(5) calculating the nitrate removal rate NO of the bacterial liquid obtained in the step (3)3 --Nrem.ratio(％)；
(6) And (4) calculating the light transmittance of the demulsified water phase by using the multiple linear regression equation, and further predicting the demulsification effect of the strain obtained in the step (3) on the ASP flooding produced water.
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