A method for determining the aerobic biodegradability of a base fluid of a drilling fluid in seawater

Abstract

The application discloses a method for determining aerobic biodegradability of seawater of a drilling fluid base fluid. The application provides a fluorescence detection method and a detection reagent suitable for detecting living bacteria ATP in seawater environment, and solves the problems of ATP unable to detect living bacteria and high salinity environment affecting enzyme activity. The application also provides a method for preparing seawater inoculation water samples, improves the existence probability of high-efficiency degradation bacteria capable of degrading the base fluid, reduces the loss of microorganism species caused by personnel operation, thereby shortens the lag phase of the base fluid, weakens the variability between parallel samples, and improves the quality of biodegradation data.

Description

Technical Field

[0001] This invention belongs to the field of offshore oil drilling, and specifically relates to a method for determining the aerobic biodegradability of seawater as a drilling fluid base. Background Technology

[0002] Non-aqueous drilling fluids are continuous-phase oil-based, mineral oil-based, or synthetic-based fluids that are insoluble in water. They offer unparalleled advantages over water-based drilling fluids in maintaining wellbore stability and improving drilling speed, and are commonly used in complex well operations such as deep wells, shale gas wells, and ultra-long horizontal well sections. However, non-aqueous drilling fluids contain a large amount of organic compound mixtures, which can impact the ecological environment, whether during formation drilling or the discharge of waste fluids and cuttings. Biodegradation is one of the most significant transformations of drilling fluids after they enter the environment. To understand the environmental fate of drilling fluids and minimize their impact, it is necessary to conduct biodegradability studies on the base fluids.

[0003] Biodegradation research methods can be divided into three categories: field trials, laboratory simulation trials, and screening trials. Field trials are conducted under realistic environmental conditions and can elucidate the real-world environmental behavior of test substances. Laboratory simulation trials and screening trials are conducted under representative conditions in a relevant environment, studying biodegradability within a laboratory setting. Due to their high cost, time-consuming nature, and complex data, field trials and laboratory simulation trials are difficult to implement in real-world research. Screening trials are simpler and more convenient.

[0004] Among them, the OECD 306 closed-bottle method is the most representative test method for studying the aerobic biodegradability of marine base fluids. This method measures the final biodegradability of the base fluid by measuring the amount of oxygen consumed in the water medium of the closed bottle. However, this method lacks a unified standard for seawater inoculum and does not specify the initial concentration range of microorganisms in the test water sample, resulting in high variability in test results between experiments. Furthermore, seawater inoculum usually has a lower bacterial concentration than freshwater inoculum, resulting in a lower content of highly efficient degrading bacteria in the base fluid and a longer lag period, leading to false positives. In addition, this method requires the simultaneous preparation of multiple closed-bottle parallel samples and specifies at least four dissolved oxygen measurements during the experiment, with at least two parallel samples measured each time. Once the closed bottle is opened for dissolved oxygen measurement, it is invalid. Therefore, this method has low data acquisition efficiency and high labor costs. For poorly soluble drilling fluid base fluids, they are generally homogenized in water by mechanical stirring. However, in actual operation, it is difficult to ensure that the concentration of drilling fluid in parallel samples is consistent, and mechanical stirring can easily break cells, thus causing intra-experimental errors and leading to large variability in test results.

[0005] Therefore, there is an urgent need for a method suitable for detecting the biodegradability of non-water-based drilling fluids in marine environments.

[0006] Plate counting is a commonly used method for microbial counting in aquatic biodegradation experiments. However, this method can only culture a portion of heterotrophic bacteria (heterotrophic bacteria account for only 0.1-1% of those in nature), and the culture time is long (2-3 days), with significant subjective influence on counting. Its efficiency and accuracy are unsatisfactory. With continuous technological advancements, instruments that can count microorganisms in water samples more efficiently and accurately have been introduced to the market, such as flow cytometers and Newton cell counters. Flow cytometry can automate the analysis of water samples in about 20 minutes, and based on the optical properties of cells, it can detect nearly 99.9% of bacteria in the water sample. Newton cell counters require only 10 seconds for a single sample measurement, and based on advanced imaging and image recognition technologies, they can accurately analyze the number of microorganisms in water samples. However, both instruments have high purchase and maintenance costs, require highly skilled operators, and the sample preparation process for flow cytometry is particularly cumbersome.

[0007] ATP fluorescence detection technology is based on the bioluminescence principle of fireflies. It utilizes firefly luciferase with D-luciferin, adenosine triphosphate (ATP), and O2 as substrates in a Mg2+ atmosphere. 2+ Under certain conditions, ATP converts chemical energy into light energy, emitting light quanta. Therefore, by adding luciferin and luciferase to a water sample and detecting the intensity of the fluorescence signal emitted when they react with ATP, the concentration of ATP in the water sample can be indirectly reflected, thereby assessing the number of microorganisms in the sample. Compared to traditional plate counting, ATP fluorescence detection technology has advantages such as speed, simplicity, high sensitivity, no culture required, and strong quantification. Compared to flow cytometry and Newtonian cell counters, ATP fluorescence detection equipment is relatively inexpensive and has the potential for widespread adoption in the field of microbial detection. Therefore, considering both detection efficiency and economics, ATP fluorescence detection technology is more suitable for application in the process of controlling the concentration of microorganisms in water samples in this invention.

[0008] Currently, there are no reports of applying ATP fluorescence detection technology to seawater biodegradability tests. ATP fluorescence detection technology cannot distinguish between ATP from live and dead bacteria, and the activity and stability of insect fluorescent proteins are affected by various environmental factors, such as temperature, pH, and ionic strength. CN 100507525C reports a rapid detection method for microorganisms in environmental water bodies, which eliminates interference from cationic, anionic, and amphoteric surfactants by adding inhibitors, but it does not distinguish the effect of freshwater and seawater salinity on protein stability. Besides water, seawater mainly consists of a large amount of salts, such as sodium chloride, potassium chloride, calcium chloride, and magnesium chloride. Sodium chloride is the most abundant, resulting in an average seawater salinity of approximately 3.5%. Under this high salinity environment, the structure and activity of insect fluorescent proteins may be affected.

[0009] Therefore, this invention provides a fluorescent detection method and reagent suitable for detecting live bacteria ATP in seawater environments, addressing the issues of live bacteria detection and high salinity environments. Combined with a tangential flow concentration method, a water sample with a certain concentration of microbial inoculation is prepared, thereby helping to improve the reliability and stability of seawater biodegradability test results. Summary of the Invention

[0010] In view of the above-mentioned problems in the prior art, the purpose of this invention is to provide an improved method for detecting the aerobic biodegradation performance of drilling fluid base fluid in a marine environment, so as to standardize the concentration and activity of seawater inoculum, improve the quality of biodegradation data, and more stably reflect the biodegradation performance of the base fluid in a marine environment.

[0011] The first objective of this invention is to provide a method for preparing seawater inoculum samples, which utilizes ATP fluorescence detection technology and tangential flow concentration technology to complete the preparation process.

[0012] A second objective of this invention is to provide the application of seawater inoculum water samples in the testing of the biodegradability of drilling fluid base fluids.

[0013] The third objective of this invention is to provide a test method for the aerobic biodegradability of marine organisms. The method involves adding a certain volume of seawater culture medium prepared with seawater inoculum water sample to a BOD bottle, mixing it with an appropriate amount of base liquid, and culturing it for 28 days under conditions of 20℃±1℃, in the dark, in a sealed environment, and with continuous stirring. The mineralization degree of the base liquid is measured by determining the amount of oxygen consumed during this process.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] The first aspect of the present invention provides an ATP extract, wherein, based on the volume of the ATP extract, the ATP extract contains 0.05 to 5.0 g / L benzalkonium bromide (Bzk) and 0.1 to 5.0 g / L alkyl glucoside.

[0016] In some embodiments, the alkyl glucosinolate comprises one or more of decyl glucosinolate, octyl glucosinolate, lauryl glucosinolate, and hexadecyl glucosinolate. As a preferred embodiment, the alkyl glucosinolate is decyl glucosinolate.

[0017] In some embodiments, the ATP extract further comprises 0.1–1 g / L magnesium sulfate (MgSO4), 0.01–0.5 g / L ethylenediaminetetraacetic acid (EDTA), and 3–10 g / L glycylglycine.

[0018] In this invention, the addition of the nonionic surfactant alkyl glucoside (APG10) to the seawater microbial ATP extract significantly improves the detection effect. Benzalkonium bromide (Bzk) is a commonly used ATP extractant; a certain concentration of Bzk can extract ATP from cells, but it has a certain inhibitory effect on luciferase activity. APG10 is generally considered a low-toxicity nonionic surfactant. When used in combination with benzalkonium bromide, it has a good synergistic effect on microbial ATP extraction, reducing the amount of Bzk added and thus reducing Bzk's inhibitory effect on the enzyme.

[0019] A second aspect of the present invention provides a composition for detecting the concentration of ATP in seawater, comprising the ATP extract and reaction reagents described in the first aspect of the present invention;

[0020] The reaction reagents include reaction reagent buffer, luciferase, and luciferin.

[0021] In some embodiments, the reaction reagent buffer comprises, by volume, 1–20 mmol / L dithiothreitol (DTT), 1–10 mmol / L magnesium sulfate (MgSO4), 0.05–0.5 g / L bovine serum albumin (BSA), 0.01–1 g / L capryloyl hydroxamic acid (CHA), 0.1–5.0 g / L cyclodextrin (CDs), and 10–50 mmol / L glycyl glycine.

[0022] In some implementations, the concentration of luciferase is 3–30 mg / L and the concentration of luciferin is 10–1000 mg / L, based on the volume of the reaction reagent buffer.

[0023] In some implementations, the concentration of luciferase is 3–20 mg / L and the concentration of luciferin is 10–500 mg / L, based on the volume of the reaction reagent buffer.

[0024] In some implementations, the concentration of luciferase is 3–10 mg / L and the concentration of luciferin is 10–100 mg / L, based on the volume of the reaction reagent buffer.

[0025] In some implementations, the concentration of luciferase is 3–10 mg / L and the concentration of luciferin is 10–30 mg / L, based on the volume of the reaction reagent buffer.

[0026] In some embodiments, the reaction reagent is prepared by the following method:

[0027] Adjust the pH of the reaction reagent buffer to 7.4–8.0, and after standing at room temperature for 24–48 hours, dissolve the luciferase and luciferin in the reaction reagent buffer to obtain the reaction reagent.

[0028] In some embodiments, after obtaining the reaction reagent, it is gently shaken and allowed to stabilize at room temperature for half an hour before use.

[0029] In some implementations, the pH of the reaction reagent buffer is adjusted using 0.1 mol / L sodium hydroxide and 0.1 mol / L hydrochloric acid.

[0030] This invention employs ATP biofluorescence detection technology to detect ATP concentration in seawater. The invention uses sterile seawater to prepare the ATP standard solution, eliminating the influence of free ATP and salinity in the seawater on the detection results. To improve the activity of insect fluorescent proteins in seawater, the ATP extraction solution and reaction reagents described in this invention are used.

[0031] A third aspect of the present invention provides a method for detecting ATP concentration in seawater, comprising:

[0032] S1. Prepare an ATP standard solution using sterile seawater or sterile NaCl solution (3.0% sterile NaCl solution, simulating the actual concentration of NaCl in seawater), add the ATP extraction solution described in the first aspect of the present invention, let stand for 1-3 minutes, then add the reaction reagent described in the second aspect of the present invention, measure the luminescence intensity, and plot the ATP standard curve.

[0033] S2. Add the ATP extraction solution described in the first aspect of the present invention to the sample to be tested, let it stand for 1 to 3 minutes, then add the reaction reagent from the second aspect of the present invention to it, measure the RLU value, and then calculate the ATP concentration in the sample to be tested according to the ATP standard curve described in step S1.

[0034] The volume ratio of the test sample, ATP extract, and reaction reagent is 1–10: 1–5: 0.5–10.

[0035] In some implementations, the volume ratio of the test sample, ATP extract, and reaction reagent is 1:1:2.

[0036] In this invention, step S2, from the moment the ATP extract is added to the sample to be tested until the RLU value is obtained, takes 3 to 10 minutes.

[0037] In an embodiment of the present invention, the sterile seawater is obtained by filtration using a 0.22 μm filter membrane (Millex disposable needle filter PES).

[0038] In some implementations, step S1 includes:

[0039] According to the method of claim 6, step S1 includes: preparing ATP standard solutions with ATP concentrations of 10⁻⁸ mol / L, 10⁻⁹ mol / L, 10⁻¹⁰ mol / L, 10⁻¹¹ mol / L, and 10⁻¹² mol / L using sterile seawater or sterile NaCl solution; taking the ATP standard solutions, adding ATP extract, gently shaking and letting stand for 1–3 min; adding reaction reagents and measuring the luminescence intensity; plotting the ATP standard curve y = ax + b with the logarithm of ATP concentration x as the abscissa and the logarithm of relative luminescence intensity (RLU) y as the ordinate; the volume ratio of ATP standard solution, ATP extract, and reaction reagents is 1–10:1–5:0.5–10.

[0040] In some implementations, step S1 includes:

[0041] Prepare a standard ATP solution using sterile seawater (e.g., dissolve 55.1 mg of disodium ATP in 100 mL of sterile seawater to obtain 10). - 3 Take 0.5 mL of 10 mol / L ATP standard solution. -3 The ATP standard solution (mol / L) was diluted sequentially with sterile seawater to obtain an ATP concentration of 10. -8 mol / L, 10 -9 mol / L, 10 -10 mol / L, 10 -11 mol / L and 10 -12 Prepare a standard solution of 1 mol / L; take 25 μL of ATP standard solution, add 25 μL of ATP extract, shake gently and let stand for 1–3 min; add 50 μL of reaction reagent and measure its luminescence intensity; plot the ATP standard curve y = ax + b with the logarithm of ATP concentration x as the abscissa and the logarithm of relative luminescence intensity (RLU) y as the ordinate, requiring R² ≥ 0.95.

[0042] In one specific implementation, step S2 includes: taking 25 μL of the sample to be tested, adding 25 μL of the ATP extraction solution, gently shaking and letting it stand for 1 to 3 minutes, adding 50 μL of the reaction reagent, measuring its RLU value, and then calculating the ATP concentration in the sample to be tested according to the standard curve.

[0043] A fourth aspect of the present invention provides a method for preparing a seawater inoculum water sample, comprising:

[0044] (1) Filter the seawater as is to obtain coarse filtered seawater;

[0045] (2) Microorganisms in coarsely filtered seawater were enriched using filtration equipment to obtain an ATP concentration of 10. -10mol / L~10 -8 A concentrated bacterial solution of seawater at a concentration of mol / L is used to obtain a seawater inoculum water sample.

[0046] In step (2), the ATP concentration is detected using the method described in the third aspect of this invention.

[0047] In some implementations, the filtration device is a tangential flow filtration membrane device.

[0048] In some implementations, the filter membrane of the tangential flow filtration membrane package device is a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm.

[0049] In this invention, a tangential flow filtration membrane device is used to perform tangential flow circulation filtration on coarse seawater. When the ATP concentration of the concentrated bacterial solution in the seawater is 10... -10 mol / L~10 -8 A seawater inoculum sample with a certain activity was obtained by applying a concentration of mol / L.

[0050] In some implementation schemes, the seawater sample is natural seawater collected from nearshore areas at a depth of 2-20m. It is then transported to the laboratory within one day under conditions of darkness, oxygenation, and a temperature not exceeding 22°C.

[0051] In some embodiments, the coarsely filtered seawater is a water sample obtained by filtering the seawater as is through filter paper, in which particulate matter, large zooplankton, phytoplankton, and other impurities are removed. In a specific embodiment of the present invention, the filter paper is a double-loop quantitative filter paper (medium speed, model 202, Cytiva, standard number No. GB / T1914-2017).

[0052] The fifth aspect of the present invention provides a seawater inoculum water sample, which is prepared by the preparation method described in the fourth aspect of the present invention.

[0053] A sixth aspect of the present invention provides a method for determining the aerobic biodegradability of seawater as a drilling fluid base fluid, comprising:

[0054] (i) Add mineral nutrient solution to the seawater inoculum water sample according to the fifth aspect of the present invention, wherein the amount of mineral nutrient solution added is 0.1% to 0.2% by volume, to obtain a seawater culture medium containing marine inoculum;

[0055] (ii) The seawater culture medium containing marine inoculum and the drilling fluid base fluid to be tested are mixed, cultured, and the oxygen consumption is detected. The biodegradation rate of the drilling fluid base fluid is then calculated.

[0056] Based on theoretical oxygen demand (ThOD), the final concentration of the drilling fluid base fluid to be tested after mixing in step (II) is 50-100 mg O2 / L.

[0057] In some implementation schemes, the mineral nutrient solution is prepared in accordance with the existing standard GB / T 30665-2014.

[0058] In some implementations, the mineral nutrient solution includes phosphate buffer, calcium chloride solution, magnesium sulfate solution, and ferric chloride solution.

[0059] In this invention, the addition amount of the mineral nutrient solution is 0.1% to 0.2% by volume, which means that the addition amount of each mineral nutrient solution is 0.1% to 0.2% by volume.

[0060] In some implementation schemes, the cultivation conditions in step (ii) are: 28 days of cultivation at 20℃±1℃ in the dark.

[0061] In some implementations, the seawater culture medium containing the marine inoculum and the drilling fluid base fluid to be tested are mixed in a BOD bottle, with the volume of the water medium in the BOD bottle accounting for 40-70% of the BOD bottle capacity.

[0062] In one embodiment of the present invention, a blank group and a test group are set up. The amount of base liquid added in the test group is calculated based on the theoretical oxygen demand of 50-100 mg / L. Since the base liquid is a sparingly soluble substance, it is directly added to the BOD bottle. The theoretical oxygen demand ThOD (mgO2 / mg oil) can be calculated based on the elemental composition and ratio of the base liquid, such as C:H:O=c:h:o, and the calculation formula is shown in formula (1):

[0063]

[0064] Where c is the proportion of carbon in the base liquid, h is the proportion of hydrogen in the base liquid, and o is the proportion of oxygen in the base liquid.

[0065] For example, the carbon-hydrogen ratio of alkane-based liquids is C:H≈n:2n+2, and the calculated ThOD≈3.46mg O2 / mg oil.

[0066] In one embodiment of the present invention, a pressure-measuring respiration meter is used to detect the oxygen consumption in the bottle, and carbon dioxide in the bottle is absorbed by flake sodium hydroxide or potassium hydroxide. When the carbon dioxide in the bottle is absorbed, the change in pressure difference in the bottle is the change in oxygen partial pressure, and the change in pressure difference can be converted into the oxygen consumption in the bottle. The percentage of biological oxygen consumption obtained after correction for microbial endogenous respiration to the theoretical oxygen demand (ThOD) of the base liquid is the biodegradation rate of the base liquid, as shown in formula (2):

[0067]

[0068] in, The total O2 content (mg) consumed by the test group samples. ThOD represents the cumulative O2 content (mg) consumed by the blank group sample, m represents the added mass of the base solution (mg), and ThOD represents the theoretical oxygen demand of the base solution (mg).

[0069] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0070] 1. The ATP fluorescence detection method for seawater inoculum of the present invention is simple to operate and fast to detect. The detection result can be obtained in just a few minutes.

[0071] The ATP concentration of concentrated bacterial solution in seawater can be determined using a standard curve. This invention provides a fluorescent detection method and reagent suitable for detecting live bacterial ATP in seawater environments, solving the problems of inability to detect live bacteria with ATP and the influence of high salinity on enzyme activity.

[0072] 2. The tangential flow concentration technology of the present invention, that is, the filtration method in which the filter membrane is placed horizontally and the feed liquid flows horizontally parallel to the filter membrane, can reduce the breakage of microbial cells and achieve a high cell recovery rate.

[0073] 3. This invention combines ATP fluorescence detection technology and tangential flow concentration technology to rapidly prepare seawater inoculated water samples with a certain bacterial concentration. This increases the probability of the presence of highly efficient degrading bacteria that can degrade the base solution, reduces the loss of microbial species due to human operation, thereby shortening the lag period of the base solution, reducing the variability between parallel samples, and improving the quality of biodegradation data.

[0074] 4. The pressure-measuring breathalyzer can monitor oxygen levels in the same sample bottle for 28 days in real time. Data collection is convenient, and there is no need to prepare a large number of parallel experiments, resulting in less variability within the experiment.

[0075] 5. This invention enables marine inoculum to have appropriate concentrations and good biological activity, reducing variability within and between laboratories, thereby improving the reliability and stability of biodegradability test results. Attached Figure Description

[0076] Figure 1 This is a flowchart illustrating the preparation process of the seawater inoculum water sample according to the present invention.

[0077] Figure 2 The effect of different concentrations of Bzk on the extraction of ATP from Pseudomonas aeruginosa;

[0078] Figure 3 The effect of mixed solutions of different concentrations of Bzk and APG10 on ATP extraction from microbial cells. Detailed Implementation

[0079] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0080] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0081] In the specification and claims, the terms "including" and "comprising" should be understood as "including, but not limited to". The specific details disclosed are for the purpose of making the present invention easier to understand. Those skilled in the art who implement this solution using one or more technical details are also considered to be implementing the technical solution of the present invention.

[0082] Unless otherwise specified, all reagents used in the examples are commercially available.

[0083] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not described in detail in this invention all adopt conventional techniques in this technical field.

[0084] Example 1

[0085] First, prepare a reaction buffer consisting of: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycylglycine, 0.5 g / L capryloyl hydroxamic acid, and 3 g / L cyclodextrin, pH 7.8. Incubate the reaction buffer at room temperature for 48 hours. Then, dissolve the luciferase and luciferin in the reaction reagent buffer to obtain a reaction reagent with a luciferase concentration of 3 mg / L and a luciferin concentration of 25 mg / L.

[0086] Prepare 10 liters of solution using 3.0% sterile NaCl solution (simulating the actual concentration of NaCl in seawater). -6Take 1 mL of mol / L ATP solution and mix it with 1 mL of Bzk solution of a certain concentration (0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L, 1.5 g / L, 2 g / L) prepared with sterile water. After gently shaking and standing for 3 min, add the reaction reagent and test the relative luminescence intensity. The effect of Bzk concentration on luciferase activity was investigated by the relative luminescence intensity. It was found that the Bzk concentration had little effect on enzyme activity when it was between 0 and 0.04%.

[0087] Add 1 mL of OD 600 ≈0.4 g of Bacillus subtilis and Pseudomonas aeruginosa were mixed with 1 mL of Bzk solutions of certain concentrations (0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1 g / L, 1.5 g / L, 2 g / L) prepared with 3.0% sterile NaCl solution. The mixture was gently shaken and allowed to stand for 3 minutes. After adding the reaction reagents, the relative luminescence intensity was measured. The effect of Bzk concentration on ATP extraction from Pseudomonas aeruginosa was investigated by measuring the relative luminescence intensity. It was found that Bzk concentrations between 0.04% and 0.06% showed better extraction effects on Pseudomonas aeruginosa (see [link to article]). Figure 2 ).

[0088] Take 1 mL of 10 -6 A mol / L ATP standard NaCl solution was mixed with 1 mL of a mixed solution containing a certain concentration of Bzk and APG10. The concentration of Bzk could be 0.1 g / L, 0.2 g / L, 0.3 g / L, or 0.4 g / L, and the concentration of APG10 could be 0.1 g / L, 0.2 g / L, 0.5 g / L, 1 g / L, 2 g / L, or 5 g / L. After gently shaking and standing for 3 min, the reaction reagent was added, and the relative luminescence intensity was measured. The effect of the mixed solution of Bzk and APG10 on luciferase activity was investigated by the relative luminescence intensity. It was found that when the concentration of Bzk was less than 0.4 g / L, the addition of 0.1 g / L to 5 g / L of APG10 did not have a significant inhibitory effect on enzyme activity.

[0089] Add 1 mL of OD 600≈0.4 g of Bacillus subtilis, Pseudomonas aeruginosa, and 1 mL of a mixed solution of Bzk and APG10 prepared with 3.0% sterile NaCl solution at a certain concentration were placed in a glass tube. The concentrations of Bzk could be 0.1 g / L, 0.2 g / L, 0.3 g / L, or 0.4 g / L, and the concentrations of APG10 could be 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, or 5 g / L. After gently shaking and standing for 3 minutes, the reaction reagents were added, and the relative luminescence intensity was measured. The relative luminescence intensity was used to investigate the extraction effect of the mixed solution of Bzk and APG10 on ATP from Bacillus subtilis or Pseudomonas aeruginosa. It was found that the combination of low concentration benzalkonium bromide and a certain amount of APG10 had a good synergistic effect on the extraction of ATP from microbial cells (see [link to study]). Figure 3 ).

[0090] Example 2: Extraction effect of ATP extract on seawater

[0091] First, prepare a reaction buffer consisting of: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycylglycine, 0.5 g / L capryloyl hydroxamic acid, and 3 g / L cyclodextrin, pH 7.8. Incubate the reaction buffer at room temperature for 48 hours. Then, dissolve the luciferase and luciferin in the reaction reagent buffer to obtain a reaction reagent with a luciferase concentration of 3 mg / L and a luciferin concentration of 25 mg / L.

[0092] The ATP extraction solution was prepared using sterile ultrapure water, and its composition is as follows:

[0093] ATP extract 1: 0.2 g / L Bzk, 2 g / L APG10;

[0094] ATP extract 2: 0.2 g / L Bzk, 2 g / L APG10, 0.5 g / L MgSO4;

[0095] ATP extract 3: 0.2 g / L Bzk, 2 g / L APG10, 0.5 g / L MgSO4, 0.5 g / L EDTA;

[0096] ATP Extract 4: 0.2 g / L Bzk, 2 g / L APG10, 0.5 g / L MgSO4, 0.5 g / L EDTA, 5 g / L glycylglycine.

[0097] After preparation, adjust the pH to 7.4 using 0.1 mol / L sodium hydroxide and 0.1 mol / L hydrochloric acid.

[0098] Take 25 μL of coarsely filtered seawater, add 25 μL of ATP extraction solution, shake gently and let stand for 1–3 min. Add 50 μL of reaction reagent and measure its luminescence intensity. The results are shown in Table 1.

[0099] Table 1. Comparison of relative luminescence intensity between ATP extract and seawater sample.

[0100] ATP extract number Relative luminous intensity (RLU) 1 16732 2 18641 3 19037 4 19844

[0101] As shown in Table 1, Mg 2+ EDTA and glycine buffer play an important role in the stability of ATP. Appropriate addition of Mg... 2++ EDTA and glycine buffer can improve the extraction efficiency of ATP.

[0102] Example 3: Effect of luciferin-luciferase reaction reagent buffer on enzyme activity

[0103] The luciferin-luciferase reaction reagent buffer solution was prepared using sterile ultrapure water, and its composition is as follows:

[0104] Buffer 1: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycyl glycine, pH = 7.4;

[0105] Buffer 2: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycyl glycine, 0.5 g / L octanoyl hydroxamic acid, pH = 7.4;

[0106] Buffer 3: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycyl glycine, 3 g / L cyclodextrin, pH = 7.4;

[0107] Buffer 4: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycyl glycine, 0.5 g / L capryloyl hydroxamic acid, 3 g / L cyclodextrin, pH = 7.4;

[0108] Buffer 5: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycyl glycine, 0.5 g / L capryloyl hydroxamic acid, 3 g / L cyclodextrin, pH = 7.8;

[0109] Buffer 6: 10 mM DTT, 5 mM MgSO4, 0.2 g / L BSA, 25 mM glycyl glycine, 0.5 g / L capryloyl hydroxamic acid, 3 g / L cyclodextrin, pH=8.

[0110] The reaction reagent buffer was incubated at room temperature for 48 hours. Then, luciferase and luciferin were dissolved in the reaction reagent buffer at a concentration of 3 mg / L and luciferin concentration of 25 mg / L. The solution was gently shaken to dissolve, and allowed to stabilize at room temperature for half an hour before use. The ATP extraction solution contained: 0.2 g / L Bzk, 2 g / L APG10, 0.5 g / L MgSO4, 0.5 g / L EDTA, and 5 g / L glycylglycine. 25 μL of coarsely filtered seawater was added to 25 μL of the ATP extraction solution, gently shaken, and allowed to stand for 3 minutes. 50 μL of the reaction reagent was added, and the luminescence intensity was measured. The results are shown in Table 2.

[0111] Table 2 Comparison of relative luminescence intensity between reaction reagents and seawater samples

[0112] Reaction reagent buffer number Relative luminous intensity (RLU) 1 28257 2 32293 3 33037 4 37840 5 39194 6 32296

[0113] As can be seen from the data in Table 2:

[0114] (1) A comparison of the effects of buffer 1 and buffer 2 shows that the addition of the chelating agent octanoyl hydroxamic acid can enhance enzyme activity. Octanoyl hydroxamic acid can selectively and efficiently chelate metal ions and is easily biodegradable. Therefore, it is often used as a chelating agent in cosmetics, mineral flotation and other fields, and is a good substitute for the traditional chelating agent disodium EDTA. Octanoyl hydroxamic acid exists in both keto and enol tautomers. The oxygen and nitrogen positions of the oxime group are close to each other and both have a pair of electrons. Therefore, it can form stable polycyclic metal chelates with various metal ions, thereby hindering the inhibitory effect of various metal ions on enzymes in seawater.

[0115] (2) A comparison of the effects of buffer 1 and buffer 3 shows that the addition of cyclodextrin can also enhance enzyme activity. Cyclodextrin is a cyclic oligosaccharide formed by α-1,4-glycosidic bonds, containing 6, 7, and 8 D-glucopyranose units, respectively called α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. The special structure of cyclodextrin gives it the characteristics of being hydrophobic inside and hydrophilic outside. Its hydrophobic cavity can bind to enzyme molecular groups to form inclusion complexes, thereby protecting the enzyme structure and active sites, reducing the degree of enzyme denaturation, and thus improving the enzyme's thermal stability.

[0116] (3) Buffer 4 is based on buffers 2 and 3, with the addition of octanoyl hydroxamic acid and cyclodextrin, which further significantly enhances the enzyme activity.

[0117] (4) Comparing the effects of buffer solutions 4, 5 and 6, it can be seen that the effect is better when the pH value of the buffer solution is 7.8.

[0118] Example 4

[0119] Seawater samples were taken from natural seawater near the Chenghai drilling platform in the Dagang Oilfield, at a depth of 2-5 meters. Under conditions of darkness, oxygenation, and a temperature not exceeding 22°C, the samples were transported to the laboratory within one day. Particulate matter, large zooplankton, phytoplankton, and other impurities were filtered from the water samples using cotton medium-speed quantitative filter paper to obtain coarsely filtered seawater.

[0120] ATP in seawater includes free ATP. free and ATP in microbial cells cell The two parts were filtered through a 0.22 μm filter membrane to obtain sterile seawater, which at this point contained only ATP. free Immediately prepare 10 with sterile seawater -6 10 mol / L ATP standard solution was prepared by serial dilution with sterile seawater. -8 mol / L, 10 -9 mol / L, 10 -10 mol / L, 10 -11 mol / L and 10 - 12 Five ATP standard solutions at concentrations of mol / L. The standard concentrations here refer only to the concentration of the added ATP chemical reagent and do not include ATP itself. free And by default, the ATP in the standard solution free The concentration was the same as in seawater. The relative luminescence intensities were measured after adding ATP extract and reaction reagents, as shown in Table 3. A standard curve was plotted, and the relationship between lgc(ATP) and lgRLU was obtained as y = 0.398x + 9.12, R... 2 =0.991.

[0121] The ATP extraction solution had the same components as ATP extraction solution 4 in Example 2. The reaction reagents were prepared using reaction buffer 5, luciferase, and luciferin from Example 3, with the luciferase concentration at 3 mg / L and the luciferin concentration at 25 mg / L.

[0122] Table 3. Comparison of ATP Concentration and Relative Luminous Intensity

[0123] ATP concentration c (mol / L) Relative luminous intensity (RLU) lgRLU <![CDATA[10 -8 ]]> 898734 5.95 <![CDATA[10 -9 ]]> 377107 5.58 <![CDATA[10 -10 ]]> 113235 5.05 <![CDATA[10 -11 ]]> 50118 4.70 <![CDATA[10 -12 ]]> 25119 4.40

[0124] The coarsely filtered seawater is circulated and filtered using a tangential flow filtration membrane device to enrich seawater microorganisms and obtain a concentrated seawater bacterial solution. The clamp area is 0.1 m². 2 Use 1-5 membrane packs, polytetrafluoroethylene filter membranes, feed flow rate 8 L / min -1 ·m -2 Discharge flow rate 2-3 L / min -1 ·m -2After tangential flow filtration for a period of time, 25 μL of the circulating seawater bacterial solution was taken, and 25 μL of ATP extraction solution was added. The mixture was gently shaken and allowed to stand for 3 minutes. Then, 50 μL of reaction reagent was added, and the RLU was measured to be 683526. Based on the standard curve, the ATP concentration of the circulating seawater bacterial solution was calculated to be 5.57 × 10⁻⁶. -9 mol / L, to obtain seawater inoculum water samples. Figure 1 This is a flowchart illustrating the preparation process of the seawater inoculum water sample according to the present invention.

[0125] The mineral nutrient solution was prepared according to the existing standard (described in section 6.4.1 of GB / T 30665-2014):

[0126] Phosphate buffer: Weigh 8.50g potassium dihydrogen phosphate (KH2PO4), 21.75g ​​dipotassium hydrogen phosphate (K2HPO4), 33.30g disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O) and 0.50g ammonium chloride (NH4Cl), dissolve in distilled water, and bring the volume to 1L.

[0127] Calcium chloride solution: Weigh 27.50g of anhydrous calcium chloride (CaCl2), dissolve it in distilled water, and bring the volume to 1L.

[0128] Magnesium sulfate solution: Weigh 22.50g of magnesium sulfate heptahydrate (MgSO4·7H2O), dissolve it in distilled water, and bring the volume to 1L.

[0129] Ferric chloride solution: Weigh 0.25g of ferric chloride hexahydrate (FeCl3·6H2O), dissolve it in distilled water, and bring the volume to 1L.

[0130] Each mineral nutrient solution (0.1% by volume) was added to the seawater inoculum sample to obtain a seawater culture medium containing the marine inoculum. 400 mL of seawater culture medium and 8 mg of alkane-based solutions were added to the BOD bottles of the experimental group, namely, a laboratory-made synthetic alkane solution (GTL1) and a commercially available gas-to-oil linear paraffin alkane solution (GTL2). The culture was carried out at 20°C in the dark for 28 days, and the oxygen consumption in the bottles was measured and recorded using a pressure-measuring respiration meter.

[0131] Example 5

[0132] Seawater samples were collected from natural seawater near Qingdao, at a depth of 10-20m. Under conditions of darkness, oxygenation, and a temperature not exceeding 22℃, the samples were transported to the laboratory within one day. Particulate matter, large zooplankton, phytoplankton, and other impurities were filtered from the water samples using cotton medium-speed quantitative filter paper to obtain coarsely filtered seawater. This coarsely filtered seawater was then circulated through a tangential flow filtration membrane device to enrich the seawater microorganisms, resulting in a concentrated seawater bacterial solution. The clamping area was 0.1m². 2Use 1-5 membrane packs, polytetrafluoroethylene filter membranes, feed flow rate 8 L / min -1 ·m -2 Discharge flow rate 2-3 L / min -1 ·m -2 .

[0133] The ATP extraction solution had the same components as ATP extraction solution 4 in Example 2. The reaction reagents were prepared using reaction buffer 5, luciferase, and luciferin from Example 3, with the luciferase concentration at 3 mg / L and the luciferin concentration at 25 mg / L.

[0134] ATP standard solution was prepared using sterile seawater and serially diluted to obtain 10. -8 mol / L, 10 -9 mol / L, 10 -10 mol / L, 10 -11 mol / L and 10 -12 Five ATP standard solutions of different concentrations (mol / L) were prepared, and the relative luminescence intensities were measured after adding ATP extract and reaction reagents. A standard curve was plotted, yielding the relationship between lgc(ATP) and lgRLU as y = 0.409x + 9.23, R... 2 =0.995.

[0135] After tangential flow filtration for a period of time, 25 μL of the seawater circulating bacterial solution was taken, and 25 μL of ATP extraction solution was added. The mixture was gently shaken and allowed to stand for 3 minutes. Then, 50 μL of reaction reagent was added, and the RLU was measured to be 429173. Based on the standard curve, the ATP concentration of the seawater circulating bacterial solution was calculated to be 1.60 × 10⁻⁶. -9 mol / L, to obtain seawater inoculum water samples.

[0136] The mineral nutrient solutions were prepared according to existing standards (same as in Example 4). Each mineral nutrient solution (0.1% volume concentration) was added to the seawater inoculum sample to obtain a seawater culture medium containing the marine inoculum. 400 mL of seawater culture medium and 8 mg of alkane-based solutions were added to the BOD bottles of the experimental groups, respectively. The alkane-based solutions were commercially available syngas-to-oil (GTL2), highly refined mineral oil (REFINED), and white oil (WO). The mixture was incubated at 20°C in the dark for 28 days, and the oxygen consumption in the bottles was measured and recorded using a pressure-measuring respiration meter.

[0137] Example 6

[0138] Seawater samples were collected from natural seawater near Ningbo, at a depth of 10-20m. Under conditions of darkness, oxygenation, and a temperature not exceeding 22℃, the samples were transported to the laboratory within one day. Particulate matter, large zooplankton, phytoplankton, and other impurities were filtered from the water samples using cotton medium-speed quantitative filter paper to obtain coarsely filtered seawater. This coarsely filtered seawater was then circulated through a tangential flow filtration membrane device to enrich the seawater microorganisms, resulting in a concentrated seawater bacterial solution. The clamping area was 0.1m². 2 Use 1-5 membrane packs, polytetrafluoroethylene filter membranes, feed flow rate 8 L / min -1 ·m -2 Discharge flow rate 2-3 L / min -1 ·m -2 .

[0139] The ATP extraction solution had the same components as ATP extraction solution 4 in Example 2. The reaction reagents were prepared using reaction buffer 5, luciferase, and luciferin from Example 3, with the luciferase concentration at 3 mg / L and the luciferin concentration at 25 mg / L.

[0140] ATP standard solution was prepared using sterile seawater and serially diluted to obtain 10. -8 mol / L, 10 -9 mol / L, 10 -10 mol / L, 10 -11 mol / L and 10 -12 The relative luminescence intensities of five ATP standard solutions at five different concentrations (mol / L) after the addition of ATP extract and reaction reagents are shown in Table 1. A standard curve was plotted, yielding the relationship between lgc(ATP) and lgRLU as y = 0.391x + 9.09, R... 2 =0.994.

[0141] After tangential flow filtration for a period of time, 25 μL of the seawater circulating bacterial solution was taken, and 25 μL of ATP extraction solution was added. The mixture was gently shaken and allowed to stand for 3 minutes. Then, 50 μL of reaction reagent was added, and the RLU was measured to be 318753. Based on the standard curve, the ATP concentration of the seawater circulating bacterial solution was calculated to be 6.72 × 10⁻⁶. -10 mol / L, to obtain seawater inoculum water samples.

[0142] The mineral nutrient solutions were prepared according to existing standards (same as in Example 4). Each mineral nutrient solution (0.1% volume concentration) was added to the seawater inoculum sample to obtain a seawater culture medium containing the marine inoculum. 400 mL of seawater culture medium and 8 mg of GTL2 or an ester-based solution were added to the BOD bottles of the experimental group, respectively. The ester-based solution was an ester base oil (EO) generated by the reaction of lauric acid and n-hexanol, with the molecular formula C. 18 H 36O2, ThOD = 2.93 mg O2 / mg oil. The cells were cultured at 20°C in the dark for 28 days, and the oxygen consumption within the bottles was measured and recorded using a pressure-based respiration meter.

[0143] Comparative Example 1

[0144] Seawater samples were collected from natural seawater near the Chenghai drilling platform in the Dagang Oilfield, at a depth of 2-5 meters. Under conditions of darkness, oxygenation, and a temperature not exceeding 22°C, the samples were transported to the laboratory within one day. Particulate matter, large zooplankton, phytoplankton, and other impurities were filtered from the water samples using cotton medium-speed quantitative filter paper to obtain coarsely filtered seawater. After serial dilution, the coarsely filtered seawater samples were used to count microorganisms using a hemocytometer and a digital microscope; the count result was 5.23 × 10⁻⁶ microorganisms. 10 Microorganisms in coarsely filtered seawater samples were counted using the seawater agar plate counting method recommended in GB / T 30665-2014. The culture medium used was 2216E marine agar, and the count result was 4.5 × 10⁻⁶ CFU / L. 6 / L. Mineral nutrient solution reference (same as Example 4). Add each mineral nutrient solution (0.1% volume concentration) to the seawater inoculum sample to obtain seawater culture medium containing marine inoculum. Add 400 mL of seawater culture medium and 8 mg of alkane base solution to the BOD bottles of the experimental group, where the alkane base solutions are GTL1 and GTL2 respectively. Incubate at 20℃ in the dark for 28 days, and detect and record the oxygen consumption in the bottle using a pressure-measuring respiration meter.

[0145] Comparative Example 2

[0146] Seawater samples were collected from natural seawater near Qingdao, at a depth of 10-20m. Under conditions of darkness, oxygenation, and a temperature not exceeding 22℃, the samples were transported to the laboratory within one day. Particulate matter, large zooplankton, phytoplankton, and other impurities were filtered from the water samples using cotton medium-speed quantitative filter paper to obtain coarsely filtered seawater. After serial dilution, the coarsely filtered seawater samples were used to count microorganisms using a hemocytometer and a digital microscope; the count result was 1.32 × 10⁻⁶ microorganisms. 10 Microorganisms in coarsely filtered seawater samples were counted using the seawater agar plate counting method recommended in GB / T 30665-2014. The culture medium used was 2216E marine agar, and the count result was 4.8 × 10⁻⁶ CFU / L. 5 / L. Mineral nutrient solution reference (same as Example 4). Add each mineral nutrient solution (0.1% volume concentration) to the seawater inoculum water sample to obtain seawater culture medium containing marine inoculum. Add 400 mL of seawater culture medium and 8 mg of GTL2 to the BOD bottles of the experimental group. Incubate at 20°C in the dark for 28 days, and detect and record the oxygen consumption in the bottle using a pressure-measuring respiration meter.

[0147] Comparative Example 3

[0148] Seawater samples were collected from natural seawater near the Chenghai drilling platform in the Dagang Oilfield, at a depth of 2-5 meters. Under conditions of darkness, oxygenation, and a temperature not exceeding 22°C, the samples were transported to the laboratory within one day. The coarsely filtered seawater was aged for 7 days at 20°C under darkness and oxygenation to remove excess dissolved organic carbon. Particulate matter, large zooplankton, phytoplankton, and other impurities were filtered from the water samples using cotton medium-speed quantitative filter paper to obtain coarsely filtered seawater. Test samples were prepared according to GB / T30665-2014. The test base solution consisted of a self-made synthetic base solution and a purchased gas-to-oil synthetic base solution, with a concentration of 2 mg / L. The samples were incubated at 20°C under darkness for 28 days. Dissolved oxygen levels in the sealed bottles were measured using a dissolved oxygen meter on days 0 and 28 to calculate the biodegradation rate.

[0149] Comparative Example 4

[0150] Seawater samples were collected from natural seawater near Qingdao at a depth of 10-20m. Under conditions of darkness, oxygenation, and a temperature not exceeding 22℃, the samples were transported to the laboratory within one day. The coarsely filtered seawater was aged for 7 days at 20℃ under darkness and oxygenation to remove excess dissolved organic carbon. Particulate matter, large zooplankton, phytoplankton, and other impurities were filtered from the water samples using cotton medium-speed quantitative filter paper to obtain coarsely filtered seawater. Test samples were prepared according to GB / T 30665-2014, with a test base solution concentration of 2 mg / L. The samples were incubated at 20℃ under darkness for 28 days. Dissolved oxygen levels in the sealed bottles were measured using a dissolved oxygen meter on days 0 and 28 to calculate the biodegradation rate.

[0151] Table 4. Results of the 28-day biodegradability test of the base liquid in Examples 4, 5, and 6.

[0152]

[0153] Table 5. Results of biodegradability test of base solutions in Comparative Examples 1–4 after 28 days.

[0154]

[0155]

[0156] The seawater inoculum prepared in Examples 4, 5, and 6 were derived from natural seawater from different regions. The ATP concentration of the seawater inoculum samples was controlled at 10 ATP using ATP fluorescence detection and tangential flow concentration techniques. -10 mol / L~10 -8 Within the mol / L range (5.57×10), -9 mol / L, 1.60×10 -9 mol / L and 6.72×10 -10(mol / L) was used for seawater biodegradation experiments. Five replicates were performed for each sample, and the standard deviation was calculated.

[0157] The seawater inoculum prepared in Comparative Examples 1 and 2 were derived from natural seawater from different regions. Only coarse filtration and microbial counting were performed on the seawater for the seawater biodegradation experiment. The microbial concentration of the seawater inoculum taken from Dagang was 5.23 × 10⁻⁶. 10 Cells / L (hemocytometer method) or 4.5 × 10⁻⁶ 6 The microbial concentration of the inoculum taken from the seawater near Qingdao was 1.32 × 10⁶ CFU / L (seawater agar plate counting method). 10 Cells / L (hemocytometer method) or 4.8 × 10⁻⁶ 5 Count / L (seawater agar plate counting method). Five replicates were performed for each sample, and the standard deviation was calculated.

[0158] The seawater inoculum prepared in Comparative Examples 3 and 4 were derived from natural seawater from different regions. The seawater underwent only aging and filtration, and was tested according to the OECD 306B (GB / T 30665-2014) method for aerobic biodegradability of seawater. Five replicates were used for each sample, and the standard deviation was calculated.

[0159] According to the biodegradation test results in Tables 4 and 5:

[0160] (1) In Examples 4, 5 and 6, the biodegradation rates of GTL2 measured using seawater microbial samples from different sources were 64.3±2.3%, 62.4±2.4% and 62.0±2.8%, respectively. The results of the three experiments were relatively similar and the standard deviation was small. However, in Comparative Examples 1 and 2, the biodegradation rates of GTL2 measured using seawater microbial samples from different sources were 46.0±6.6% and 56.4±5.5%, respectively. The results of the experiments were significantly different and the standard deviation was large. This indicates that the method of the present invention, by using ATP fluorescence detection technology and tangential flow concentration technology to control the concentration of seawater inoculum, improves the accuracy and stability of the biodegradability test results of alkane-based liquids. Seawater inoculums that do not use the method of the present invention, due to different sources and inconsistent concentrations, do not show stable biodegradability of alkane-based liquids.

[0161] (2) The biodegradation rates of GTL1 and GTL2 obtained by using the same source of seawater microorganisms but different inoculum preparation methods in Example 4 and Comparative Example 1 are different. After the seawater was concentrated in Example 4, the concentration of seawater inoculum increased, the probability of the presence of highly efficient degrading bacteria in the base liquid increased, and the biodegradation ability of the test base liquid was enhanced. The biodegradation rate of GTL1 and GTL2 can reach more than 60%. However, the concentration of seawater inoculum in Comparative Example 1, which was not concentrated, was small, and the survival probability of highly efficient degrading bacteria was small. Therefore, the biodegradation rate of GTL1 and GTL2 was low.

[0162] (3) Comparative Examples 3 and 4 used the OECD306B closed bottle method to test base solutions such as GTL1, GTL2, REFINED, and WO. The measured biodegradation rates were low and the standard deviations were large, indicating poor parallelism. In particular, white oil WO contained more toxic substances compared with other tested base solutions, which was detrimental to the biodegradation process. Therefore, concentration can improve the survival rate of highly efficient degrading bacteria and help stabilize the test results. In contrast, the method of the present invention was used to test base solutions such as GTL1, GTL2, REFINED, WO, and EO in the examples. It can be seen that the measured standard deviations of biodegradation rates were small and the parallelism was good. This indicates that the method improves the accuracy and reliability of the results and is applicable to most alkane and ester base solutions.

[0163] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An ATP extract, wherein, Based on the volume of the ATP extract, the ATP extract contains 0.05–5.0 g / L benzalkonium bromide and 0.1–5.0 g / L alkyl glucoside.

2. The ATP extract according to claim 1, wherein, The alkyl glucoside comprises one or more of decyl glucoside, octyl glucoside, lauryl glucoside, and hexadecyl glucoside.

3. The ATP extract according to claim 1 or 2, wherein, The ATP extract also contains 0.1–1 g / L magnesium sulfate, 0.01–0.5 g / L ethylenediaminetetraacetic acid, and 3–10 g / L glycylglycine.

4. A composition for detecting ATP concentration in seawater, comprising the ATP extract and reaction reagent as described in any one of claims 1-3; The reaction reagents include reaction reagent buffer, luciferase, and luciferin.

5. The composition according to claim 4, wherein, The reaction reagent buffer contains, by volume, 1–20 mmol / L dithiothreitol, 1–10 mmol / L magnesium sulfate, 0.05–0.5 g / L bovine serum albumin, 0.01–1 g / L capryloyl hydroxamic acid, 0.1–5.0 g / L cyclodextrin, and 10–50 mmol / L glycyl glycine.

6. The composition according to claim 4, wherein, The concentration of luciferase is 3–30 mg / L and the concentration of luciferin is 10–1000 mg / L, based on the volume of the reaction reagent buffer.

7. The composition according to any one of claims 4-6, wherein, The concentration of luciferase is 3–10 mg / L and the concentration of luciferin is 10–30 mg / L, based on the volume of the reaction reagent buffer.

8. A method for detecting ATP concentration in seawater, comprising: S1. Prepare an ATP standard solution using sterile seawater or sterile NaCl solution, add the ATP extract according to any one of claims 1-3, let stand for 1-3 min, then add the reaction reagent according to any one of claims 4-7, measure the luminescence intensity, and plot the ATP standard curve; S2. Add the ATP extraction solution according to any one of claims 1-3 to the sample to be tested, let it stand for 1-3 min, then add the reaction reagent according to any one of claims 4-7, measure the luminescence intensity, and then calculate the ATP concentration in the sample to be tested according to the ATP standard curve described in step S1. The volume ratio of the test sample, ATP extract, and reaction reagent is 1–10: 1–5: 0.5–10.

9. A method for preparing a seawater inoculum water sample, comprising: (1) Filter the seawater as is to obtain coarse filtered seawater; (2) Microorganisms in coarsely filtered seawater were enriched using filtration equipment to obtain an ATP concentration of 10. -10 mol / L~10 - 8 A concentrated bacterial solution of seawater at a concentration of mol / L is used to obtain a seawater inoculum water sample. In step (2), the ATP concentration is detected using the method described in claim 8.

10. The preparation method according to claim 9, wherein, The filtration equipment is a tangential flow filtration membrane device.

11. The preparation method according to claim 10, wherein, The filter membrane of the tangential flow filtration membrane packaging device is a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm.

12. A seawater inoculum sample, which is prepared by the preparation method according to any one of claims 9-11.

13. A method for determining the aerobic biodegradability of seawater as a drilling fluid base fluid, comprising: (i) Add mineral nutrient solution to the seawater inoculum water sample as described in claim 12, wherein the amount of mineral nutrient solution added is 0.1% to 0.2% by volume, to obtain a seawater culture medium containing marine inoculum; (ii) The seawater culture medium containing marine inoculum and the drilling fluid base fluid to be tested are mixed, cultured, and the oxygen consumption is detected. The biodegradation rate of the drilling fluid base fluid is then calculated. Based on theoretical oxygen demand, the final concentration of the drilling fluid base fluid to be tested after mixing in step (II) is 50-100 mg O2 / L.

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