A method for rapidly detecting microbial concentration in wastewater samples containing heavy oil using a UV-visible spectrophotometer

Abstract

The present invention provides a method for quickly detecting the concentration of microorganisms in a sample of viscous oil-containing wastewater using an ultraviolet-visible spectrophotometer, which belongs to the technical field of oilfield wastewater detection. The detection method is to allow the viscous oil-containing wastewater to stand for stratification, add a surfactant, ultrasonically vibrate, centrifuge, and filter, and then measure the absorbance at wavelengths of 230nm, 320nm, 340nm, 600nm, 620nm, and 650nm. When the judgment conditions of the absorbance value are met at the same time, the judgment data is valid; the OD600nm, OD620nm, and OD650nm in the valid data are compared with the three established standard microorganism concentration-absorbance plates, and the microbial concentration value of the viscous oil-containing wastewater sample to be tested is read out and the average is taken. This detection method can eliminate the influence of various impurities in the viscous oil wastewater sample on the test results to the greatest extent, and is simple to operate, economical and efficient, highly timely, with small errors, and high reproducibility.

Description

Technical Field

[0001] The invention relates to the technical field of oilfield sewage detection, and in particular to a method for rapidly detecting the concentration of microorganisms in a sewage sample containing thick oil by using an ultraviolet-visible spectrophotometer. Background Art

[0002] Microorganisms present in oil well produced fluids are of significant significance to fields such as reservoir evolution, geochemistry, environmental geology, crude oil remediation, and microbial enhanced oil recovery. However, current technologies for measuring microbial concentrations in oily wastewater are still in their infancy and suffer from significant shortcomings. Genetic analysis methods are costly, complex, and time-consuming; test bottle techniques are significantly affected by impurities in heavy oil, resulting in large errors and poor reproducibility; and microscopic examination methods are subject to high technician error and are significantly affected by impurities. While UV-visible spectrophotometers are widely used to measure the concentration of pure bacterial solutions, the presence of heavy oil, chemicals, and particulate impurities in heavy oil wastewater not only directly affects absorbance and causes errors, but also adsorbs microorganisms, reducing microbial concentrations. Despite these limitations, UV-visible spectrophotometry offers advantages such as high accuracy, reproducibility, low cost, and convenience, making it irreplaceable by other methods. Therefore, how to use UV-visible spectrophotometer to quickly, conveniently, accurately and cheaply measure the concentration of microorganisms in heavy oil-containing wastewater samples is a technical problem that needs to be solved urgently in the industry. Summary of the Invention

[0003] The object of the present invention is to provide a method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer, so as to solve the above technical problems.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for rapidly detecting the concentration of microorganisms in a wastewater sample containing heavy oil using an ultraviolet-visible spectrophotometer, comprising the following steps:

[0006] 1) After the heavy oil wastewater sample is allowed to stand for stratification, the lower aqueous phase (A mL) is taken and a demulsifier (B mL) having a mass concentration of X% is added according to the heavy oil concentration (M%) in the heavy oil wastewater sample;

[0007] 2) subjecting the heavy oil wastewater sample to which the demulsifier was added in step 1) to sequential shaking for Tmin and centrifugation to obtain a lower aqueous phase, filtering particulate pollutants in the lower aqueous phase to obtain a filtered aqueous phase;

[0008] 3) Pour the filtered aqueous phase into a cuvette and measure the absorbance at wavelengths of 230 nm, 320 nm, 340 nm, 600 nm, 620 nm, and 650 nm. Repeat three times and take the average value, which will be recorded as OD230, OD320, OD340, OD600, OD620, and OD650, respectively.

[0009] 4) When the test results simultaneously meet OD230≤0.100, OD320≤0.100 and OD340≤0.100, it is a valid data set; if the above conditions are not met, the filtered aqueous phase is repeated in step 2);

[0010] 5) Prepare standard bacterial solutions and measure the absorbance at 600nm, 620nm and 650nm of the standard bacterial solutions at different concentrations, which are recorded as OD600 标 OD620 标 OD650 标 , plot C-OD600 with bacterial concentration as the horizontal axis and absorbance as the vertical axis. 标 、C-OD620 标 and C-OD650 标 Standard templates;

[0011] 6) The OD600, OD620 and OD650 obtained in step 3) were respectively measured on C-OD600. 标 、C-OD620 标 and C-OD650 标 Read the concentration of the bacterial solution to be tested from the standard template and record it as C 600 、C 620 and C 650 The average value of the three is the microbial concentration of the heavy oil wastewater sample.

[0012] Furthermore, the relationship between the concentration of heavy oil M%, the lower aqueous phase AmL, the mass concentration of demulsifier X%, and the volume of demulsifier BmL is as follows:

[0013]

[0014]

[0015] Furthermore, in the step 2), the rotation speed of the centrifugal treatment is 10000-14000 r / min, the centrifugal treatment time is 2 min, and the volume of the lower aqueous phase is D mL, D≥30.

[0016] Furthermore, in step 3), the amount of the filtered aqueous phase added is 3 mL, or 2 / 3 to 3 / 4 of the total volume of the cuvette.

[0017] Furthermore, in step 5), the specific steps of preparing the standard bacterial solution are: using a standard bacterial solution at the end of the logarithmic growth phase with a known concentration, adding ultrapure water to dilute it into bacterial solutions containing 0.1%, 1%, 10%, 20%, 30%, 45%, and 100%.

[0018] Furthermore, the demulsifier comprises an oil-soluble demulsifier.

[0019] Furthermore, in the step 2), the particulate pollutants in the lower aqueous phase are filtered using a 50 μm membrane.

[0020] Beneficial effects of the present invention:

[0021] The detection method of the present invention can eliminate the influence of various impurities in the viscous oily wastewater sample on the detection result to the greatest extent, and is simple to operate, economical and efficient, highly timely, with small errors and high reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the water layer of the heavy oil used in the embodiment of the present invention being severely emulsified after standing and stratifying;

[0023] Figure 2 C-OD600 prepared in the embodiment of the present invention 标 Template curve graph;

[0024] Figure 3 C-OD620 prepared in the embodiment of the present invention 标 Template curve graph;

[0025] Figure 4 C-OD650 prepared in the embodiment of the present invention 标 Template curve graph. DETAILED DESCRIPTION

[0026] The present invention provides a method for rapidly detecting the concentration of microorganisms in a wastewater sample containing heavy oil using an ultraviolet-visible spectrophotometer, comprising the following steps:

[0027] 1) After the heavy oil wastewater sample is allowed to stand for stratification, the lower aqueous phase (A mL) is taken and a demulsifier (B mL) having a mass concentration of X% is added according to the heavy oil concentration (M%) in the heavy oil wastewater sample;

[0028] 2) subjecting the heavy oil wastewater sample to which the demulsifier was added in step 1) to sequential shaking for Tmin and centrifugation to obtain a lower aqueous phase, filtering particulate pollutants in the lower aqueous phase to obtain a filtered aqueous phase;

[0029] 3) Pour the filtered aqueous phase into a cuvette and measure the absorbance at wavelengths of 230 nm, 320 nm, 340 nm, 600 nm, 620 nm, and 650 nm. Repeat three times and take the average value, which will be recorded as OD230, OD320, OD340, OD600, OD620, and OD650, respectively.

[0030] 4) When the test results simultaneously meet OD230≤0.100, OD320≤0.100 and OD340≤0.100, it is a valid data set; if the above conditions are not met, the filtered aqueous phase is repeated in step 2);

[0031] 5) Prepare standard bacterial solutions and measure the absorbance at 600nm, 620nm and 650nm of the standard bacterial solutions at different concentrations, which are recorded as OD600 标 OD620 标 OD650 标 , plot C-OD600 with bacterial concentration as the horizontal axis and absorbance as the vertical axis. 标 、C-OD620 标 and C-OD650 标 Standard templates;

[0032] 6) The OD600, OD620 and OD650 obtained in step 3) were respectively measured on C-OD600. 标 、C-OD620 标 and C-OD650 标 Read the concentration of the bacterial solution to be tested from the standard template and record it as C 600 、C 620 and C 650 The average value of the three is the microbial concentration of the heavy oil wastewater sample.

[0033] In the present invention, the relationship between the concentration of heavy oil M%, the lower aqueous phase AmL, the mass concentration of demulsifier X%, and the volume of demulsifier BmL is as follows:

[0034]

[0035]

[0036] In the present invention, the rotation speed of the centrifugal treatment in step 2) is 10,000 to 14,000 r / min, preferably 12,000 r / min; the centrifugal treatment time is 2 min, and the volume of the lower aqueous phase is D mL, where D ≥ 30.

[0037] In the present invention, in step 3), the amount of the filtered aqueous phase added is 3 mL, or 2 / 3 to 3 / 4 of the total volume of the cuvette.

[0038] In the present invention, in step 5), the specific steps of preparing the standard bacterial solution are: using a standard bacterial solution at the end of the logarithmic growth phase with a known concentration, adding ultrapure water to dilute it into bacterial solutions containing 0.1%, 1%, 10%, 20%, 30%, 45%, and 100%.

[0039] In the present invention, the demulsifier comprises an oil-soluble demulsifier, preferably a polyether demulsifier.

[0040] In the present invention, in step 2), the particulate pollutants in the lower aqueous phase are filtered using a 50 μm membrane filter, and the process can be repeated multiple times depending on the filtering effect.

[0041] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] Take the measurement of microbial concentration in the produced fluid of production well Z in block Y of the Liaohe Oilfield heavy oil reservoir as an example.

[0044] 1. Take the produced fluid from well Z and let it stand for 2.5 hours. It is observed that there is obvious stratification. The lower layer is seriously emulsified (such as Figure 1 ), the lower aqueous phase contains heavy oil at a concentration of about 75<M≤80, and 250 mL of the lower aqueous phase is taken according to Table 1.

[0045] Table 1 A, B, X, T values ​​for microbial detection of heavy oil wastewater samples

[0046] M(％) A(mL) B (mL) X(％) T(min) M≤5 ≥52 0.05 5.20 4 5＜M≤10 ≥55 0.05 11.00 6 10＜M≤15 ≥58 0.05 17.40 8 15＜M≤20 ≥62 0.06 20.67 10 20＜M≤25 ≥66 0.06 27.50 12 25＜M≤30 ≥71 0.07 30.43 14 30＜M≤35 ≥76 0.07 38.00 16 35＜M≤40 ≥83 0.08 41.50 18 40＜M≤45 ≥90 0.09 45.00 20 45＜M≤50 ≥100 0.10 50.00 22 50＜M≤55 ≥111 0.11 55.50 24 55＜M≤60 ≥125 0.12 62.50 26 60＜M≤65 ≥142 0.14 65.93 28 65＜M≤70 ≥166 0.16 72.63 30 70＜M≤75 ≥200 0.20 75.00 32 75＜M≤80 ≥250 0.25 80.00 34 80＜M≤85 ≥333 0.33 85.77 36 85＜M≤90 ≥500 0.50 90.00 38 90＜M≤95 ≥999 0.99 95.86 40 95＜M≤99 ≥5000 5.00 100.00 42

[0047] 2. Referring to Table 1, 0.25 mL of an oil-soluble polyether demulsifier with a mass concentration of 80% was added and ultrasonically shaken for 34 minutes.

[0048] 3. Centrifuge the shaken aqueous phase in a high-speed centrifuge at 12,000 rpm for 2 minutes and remove 30 mL of the lower aqueous phase.

[0049] 4. The aqueous phase after centrifugation was subjected to membrane filtration (50 μm) to remove particulate contaminants in the aqueous phase, and filtered twice.

[0050] 5. Take 3 mL of the filtered aqueous phase and drop it into a cuvette. Measure the absorbance at wavelengths of 230 nm, 320 nm, 340 nm, 600 nm, 620 nm, and 650 nm. Repeat the measurement three times and take the average value, which is recorded as OD230, OD320, OD340, OD600, OD620, and OD650, respectively. The data results are shown in Table 2 below:

[0051] Table 2 Test results

[0052]

[0053]

[0054] 6. OD230 is 0.090, OD320 is 0.081, and OD340 is 0.097. OD230≤0.100, OD320≤0.100, and OD340≤0.100 are all satisfied, so this set of data is valid.

[0055] 7. Use a known concentration of 10 6 The standard bacterial solution of cells / mL was diluted with ultrapure water to become bacterial solutions containing 0.1%, 1%, 10%, 20%, 30%, 45% and 100% respectively.

[0056] 8. Measure the OD600 at 600nm (OD600) of the 7 concentrations of the standard bacterial solution in the above steps. 标 )、620nm(OD620 标 ) and 650nm(OD650 标 ) and plot the microbial concentration C and OD600 respectively. 标 OD620 标 and OD650 标 The template is attached. Figures 2-4 .

[0057] 9. The OD600, OD620 and OD650 in the valid data are 0.097, 0.101 and 0.101 respectively. 标 、C-OD620 标 and C-OD650 标 The bacterial concentration value C read from the template 600 62994 / mL, C 620 69731 cells / mL and C 650 The average value of the three is 63734 / mL, so the microbial concentration of this heavy oil wastewater sample is 6.4×10 4 pieces / mL.

[0058] As demonstrated in the above examples, the present invention provides a method for rapidly detecting microbial concentrations in oily wastewater samples using a UV-visible spectrophotometer. This method minimizes the impact of various impurities in oily wastewater samples on test results, and is simple to operate, cost-effective, time-efficient, with minimal error and high reproducibility.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer, characterized in that: The following steps are involved: 1) After the heavy oil wastewater sample is allowed to stand for stratification, the lower aqueous phase (A mL) is taken and a demulsifier (B mL) having a mass concentration of X% is added according to the heavy oil concentration (M%) in the heavy oil wastewater sample; 2) subjecting the heavy oil wastewater sample to which the demulsifier was added in step 1) to sequential shaking for Tmin and centrifugation to obtain a lower aqueous phase, filtering particulate pollutants in the lower aqueous phase to obtain a filtered aqueous phase; 3) Pour the filtered aqueous phase into a cuvette and measure the absorbance at wavelengths of 230 nm, 320 nm, 340 nm, 600 nm, 620 nm, and 650 nm. Repeat three times and take the average value, which will be recorded as OD230, OD320, OD340, OD600, OD620, and OD650, respectively. 4) When the test results simultaneously meet OD230≤0.100, OD320≤0.100 and OD340≤0.100, it is a valid data set; if the above conditions are not met, the filtered aqueous phase is repeated in step 2); 5) Prepare standard bacterial solutions and measure the absorbance at 600nm, 620nm and 650nm of the standard bacterial solutions at different concentrations, respectively, and record them as OD600 标 OD620 标 OD650 标 , plot C-OD600 with bacterial concentration as the horizontal axis and absorbance as the vertical axis. 标 、C-OD620 标 and C-OD650 标 Standard templates; 6) The OD600, OD620 and OD650 obtained in step 3) were respectively measured on C-OD600. 标 、C-OD620 标 and C-OD650 标 Read the concentration of the bacterial solution to be tested from the standard template and record it as C 600 、C 620 and C 650， The average value of the three is the microbial concentration of the heavy oil wastewater sample.

2. The method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer according to claim 1, wherein: The relationship between the values ​​of the heavy oil concentration M%, the lower water phase AmL, the demulsifier mass concentration X%, and the demulsifier volume BmL is as follows:

3. The method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer according to claim 1 or 2, characterized in that: The rotation speed of the centrifugal treatment in the step 2) is 10000-14000 r / min, the centrifugal treatment time is 2 min, and the volume of the lower aqueous phase is D mL, D≥30.

4. The method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer according to claim 1, wherein: In the step 3), the amount of the filtered aqueous phase added is 3 mL, or 2 / 3 to 3 / 4 of the total volume of the cuvette.

5. The method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer according to claim 1, 2 or 4, wherein: In step 5), the specific steps of preparing the standard bacterial solution are: using a standard bacterial solution at the end of the logarithmic growth phase with a known concentration, adding ultrapure water to dilute it into bacterial solutions containing 0.1%, 1%, 10%, 20%, 30%, 45%, and 100%.

6. The method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer according to claim 5, characterized in that: The demulsifier comprises an oil-soluble demulsifier.

7. The method for rapidly detecting the concentration of microorganisms in a sample of wastewater containing heavy oil using an ultraviolet-visible spectrophotometer according to claim 1, wherein: In the step 2), the particulate pollutants in the lower aqueous phase are filtered using a 50 μm membrane.

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