In-situ detection kit for oil spill degradation intermediates and methods of use thereof

Abstract

The application discloses an in-situ detection kit for oil spill degradation intermediate products and a use method thereof, and belongs to the field of environmental monitoring. The kit comprises an alkane degradation indication module, an aromatic hydrocarbon degradation indication module and a total petroleum hydrocarbon residue detection module. The alkane degradation indication module utilizes fatty alcohol dehydrogenase to catalyze the reaction of fatty alcohol and NAD, NADH reduces tetrazolium salt to generate purple formazan compound for colorimetric detection. The aromatic hydrocarbon degradation indication module utilizes catechol dioxygenase to catalyze the ring opening of catechol to generate yellow semialdehyde compound for colorimetric detection. The total petroleum hydrocarbon residue detection module utilizes fluorescein-labeled cyclodextrin to form an inclusion compound with petroleum hydrocarbon to cause fluorescence quenching for fluorescence detection. The kit is matched with a portable photometer and data processing software. The detection can be completed within 15 minutes on site, and the oil spill degradation efficiency evaluation index can be calculated. The detection sensitivity reaches 5 mg / L. The kit provides a rapid screening tool for oil spill bioremediation engineering.

Description

Technical Field

[0001] This invention belongs to the field of environmental monitoring and bioremediation effect evaluation, specifically involving a rapid detection kit for petroleum hydrocarbon degradation intermediates based on colorimetric and fluorescence dual signal responses and its usage method, which is applicable to on-site process monitoring and endpoint determination in oil spill bioremediation projects. Background Technology

[0002] Marine oil spills are among the most serious environmental disasters globally, with approximately 1.2 to 1.5 million tons of oil entering the marine environment annually through various pathways. Bioremediation technology, which utilizes the metabolic capabilities of microorganisms to degrade petroleum hydrocarbons, is an important means of treating oil spill pollution. However, the accurate evaluation of the effectiveness of bioremediation has remained a key bottleneck restricting the engineering application of this technology.

[0003] Existing methods for evaluating the biodegradation of oil spills mainly include the following categories:

[0004] The first category is analytical methods based on changes in total petroleum hydrocarbon content. These methods employ large instruments such as gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to determine the residual amount of petroleum hydrocarbons in samples. While these methods offer high accuracy, they have the following drawbacks: the equipment is expensive, with a single unit costing between 500,000 and 2 million yuan; the analysis cycle is long, taking 3-7 days from sampling to report generation; and they require specialized technical personnel to operate, making rapid on-site monitoring impossible.

[0005] The second category is indirect evaluation methods based on microbial activity indicators. These include measuring changes in parameters such as chemical oxygen demand (COD), biological oxygen demand (BOD), and total organic carbon (TOC) in activated sludge. For example, the method disclosed in Chinese invention CN104111294A uses activated sludge to cultivate microorganisms over a test period of 14-50 days, indirectly evaluating the biodegradability of organic chemicals by measuring changes in dissolved oxygen, pH, and TOC. The limitations of this type of method are: the test period is too long to reflect the degradation process in real time; the correlation between indirect indicators and the actual degree of degradation is limited; and it still requires analysis using instruments such as high-performance liquid chromatography (HPLC), making the operation complex.

[0006] The third category is screening methods based on redox indicators. For example, 2,6-dichlorophenolindophenol is used to screen petroleum hydrocarbon-degrading bacteria, judging degradation activity by the color change from blue to colorless. However, this method can only qualitatively determine whether degradation activity exists, cannot quantitatively assess the degree of degradation, and cannot distinguish between alkane degradation and aromatic hydrocarbon degradation.

[0007] The microbial degradation of petroleum hydrocarbons follows specific metabolic pathways. n-Alkanes are first terminally hydroxylated by alkane monooxygenases to form fatty alcohols, which are then oxidized to fatty aldehydes by fatty alcohol dehydrogenases, and finally mineralized via β-oxidation into the tricarboxylic acid cycle. Polycyclic aromatic hydrocarbons are catalyzed by dioxygenases to form cis-dihydrodiols, which are further oxidized to catechols, and then ring-opened by catechol dioxygenases to form hemialdehydes. These degradation intermediates are direct evidence of active microbial metabolism, but rapid on-site detection methods for these intermediates are currently lacking.

[0008] In summary, existing technologies face the following pressing problems: first, long detection cycles prevent real-time on-site monitoring; second, reliance on large-scale instruments and equipment leads to high costs; third, a lack of specific detection methods for degradation intermediates; and fourth, an inability to distinguish the degradation contributions of different types of petroleum hydrocarbons. Therefore, there is an urgent need to develop an in-situ detection technology for oil spill degradation intermediates that is easy to operate, has a rapid response time, and is cost-effective, providing an effective process monitoring tool for bioremediation projects. Summary of the Invention

[0009] To address the technical problems of existing technologies that rely on gas chromatography analysis for evaluating the effectiveness of oil spill bioremediation, which involves long detection cycles, high costs, and the inability to conduct real-time on-site monitoring, this invention provides an in-situ detection kit for intermediate products of oil spill degradation and its usage method.

[0010] The first objective of this invention is to provide an in-situ detection kit for oil spill degradation intermediates. This kit targets alkane degradation intermediates, aromatic degradation intermediates, and total petroleum hydrocarbon residues through three functional modules, and employs a dual-signal response mechanism of colorimetry and fluorescence to achieve rapid on-site evaluation of oil spill degradation efficiency.

[0011] The second objective of this invention is to provide a method for using the above-mentioned kit, including a complete process of sample pretreatment, detection operation, and result interpretation.

[0012] To achieve the above objectives, the present invention adopts the following technical solution:

[0013] The in-situ detection kit for intermediate products of oil spill degradation includes an alkane degradation indicator module, an aromatics degradation indicator module, a total petroleum hydrocarbon residue detection module, and a matching portable photometer and data processing software.

[0014] The alkane degradation indicator module includes lyophilized fatty alcohol dehydrogenase powder and coenzyme. Stock solution and tetrazolium salt colorimetric solution; the fatty alcohol dehydrogenase lyophilized powder is prepared by lyophilization after purification of long-chain fatty alcohol dehydrogenase expressed by recombinant Escherichia coli, with enzyme activity not less than The coenzyme The concentration of the stock solution is pH value The concentration of tetrazolium salt in the tetrazolium salt colorimetric solution is: The tetrazolium salt is selected from one or more of 2,3,5-triphenyltetrazolium chloride, iodonitroxytetrazolium violet, or nitrobluetetrazolium.

[0015] The detection principle of the alkane degradation indicator module is as follows: fatty alcohol dehydrogenase catalyzes the oxidation of alkane terminus products fatty alcohol and coenzyme. The reaction produces reduced coenzyme NADH. NADH acts as an electron donor, reducing tetrazolium salt to produce a purple formazan compound. The color intensity is positively correlated with the concentration of fatty alcohol. The color can be semi-quantitatively interpreted using a colorimetric card or a portable photometer. The accumulation of intermediate products of alkane degradation was quantitatively determined by measuring absorbance.

[0016] The aromatic hydrocarbon degradation indicator module includes catechol dioxygenase lyophilized powder. Activation solution and reaction buffer; the lyophilized catechol dioxygenase powder is prepared by lyophilization after purification of catechol 2,3-dioxygenase from recombinant Pseudomonas aeruginosa, with an enzyme activity of not less than The The activation solution contains Ferrous ammonium sulfate and Ascorbic acid; the reaction buffer solution has a pH value of Phosphate buffer or Tris-HCl buffer, concentration of .

[0017] The detection principle of the aromatic hydrocarbon degradation indicator module is as follows: catechol dioxygenase catalyzes the reaction of catechol, a ring-opening product of polycyclic aromatic hydrocarbons, with molecular oxygen to generate a yellow 2-hydroxymucin semialdehyde compound. Changes in absorbance reflect the activity level of aromatic hydrocarbon metabolism.

[0018] The total petroleum hydrocarbon residue detection module includes a fluorescein-labeled cyclodextrin probe solution and a fluorescence stabilizer; the fluorescein-labeled cyclodextrin probe is a β-cyclodextrin or hydroxypropyl-β-cyclodextrin covalently modified with fluorescein isothiocyanate, with a modification degree of [insert degree here]. Each cyclodextrin molecule contains one luciferin molecule, and the probe concentration is [missing value]. The fluorescence stabilizer contains antioxidants and The solubilizer.

[0019] The detection principle of the total petroleum hydrocarbon residue detection module is as follows: the hydrophobic cavity of the fluorescein-labeled cyclodextrin molecule can form a host-guest inclusion complex with the hydrophobic components of petroleum hydrocarbons, causing quenching of the fluorescein emission spectrum. The percentage of fluorescence quenching is related to the residual oil concentration. The relationship is linear within the range.

[0020] The portable photometer integrates a light-emitting diode (LED) light source, a photodiode detector, and a microprocessor, and can... Absorbance was measured within the wavelength range, and a suitable configuration was prepared. Excitation light source and Emission filters are used for fluorescence detection.

[0021] The data processing software is pre-set with standard curves for alkane degradation index, aromatic degradation index and residual oil concentration, and can automatically calculate degradation efficiency evaluation index based on detection signals.

[0022] Furthermore, the alkane degradation indicator module also includes a carbon chain length selective regulator, wherein the carbon chain length selective regulator is selected from one or more combinations of polyethylene glycol monomethyl ether, Tween-80, or Span-20, and the concentration is [missing information]. It is used to regulate the substrate selectivity of fatty alcohol dehydrogenase for fatty alcohols of different carbon chain lengths.

[0023] Furthermore, the aromatic hydrocarbon degradation indicator module also includes an aromatic ring number selectivity regulator, which is selected from one or more combinations of methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin, at a concentration of [missing information]. It is used to regulate the substrate selectivity of catechol dioxygenase for aromatic hydrocarbon metabolites with different ring numbers.

[0024] Furthermore, the fluorescein-labeled cyclodextrin probe in the total petroleum hydrocarbon residue detection module is also compounded with a hydrophobic enhancer. The hydrophobic enhancer is selected from one or more of sodium cholate, sodium deoxycholate, or sodium chenodeoxycholate, and the compounding ratio is the molar ratio of cyclodextrin to the hydrophobic enhancer. .

[0025] Furthermore, the kit also includes a sample pretreatment module, which comprises centrifuge tubes, a filter membrane, and a dilution buffer; the pore size of the filter membrane is [not specified]. The dilution buffer contains... Phosphate buffer solution containing nonionic surfactants.

[0026] Furthermore, the kit also includes a standard module containing n-dodecanol standard, catechol standard, and crude oil standard, for establishing standard curves and quality control.

[0027] Furthermore, the kit also includes a colorimetric card printed with color gradients corresponding to different concentrations of degradation intermediates for semi-quantitative interpretation without instruments.

[0028] This invention also provides a method for using the above-mentioned reagent kit, including the following steps:

[0029] Step 1: Sample Collection and Pretreatment: Seawater or interstitial water samples were collected at the oil spill bioremediation site and then pretreated. Suspended particulate matter is removed by membrane filtration. If necessary, the sample concentration is adjusted to the detection range using dilution buffer.

[0030] Step 2, Alkane Degradation Indicator Module Detection: Take... The pretreated sample was added to a detection tube pre-filled with fatty alcohol dehydrogenase lyophilized powder, and then... Coenzyme Stock solution and Tetrazazole salt colorimetric solution, in reaction under conditions Observe the color changes or use a portable photometer. The absorbance was measured at the analyte, and the alkane degradation index was calculated.

[0031] Step 3: Aromatic hydrocarbon degradation indicator module detection: Take... The pretreated sample was added to a detection tube pre-filled with catechol dioxygenase lyophilized powder, and then... Activation solution and reaction buffer, in reaction under conditions Observe the color changes or use a portable photometer. The absorbance was measured to calculate the aromatic hydrocarbon degradation index.

[0032] Step 4, Total Petroleum Hydrocarbon Residue Detection: Take... The pretreated sample was added to a detection tube pre-loaded with a fluorescein-labeled cyclodextrin probe solution and vortexed to mix. Using a portable photometer excitation, Fluorescence intensity was measured under emission conditions, and residual oil concentration was calculated based on the percentage of fluorescence quenching.

[0033] Step 5: Comprehensive Evaluation: Based on the algorithm preset in the data processing software, the oil spill degradation efficiency evaluation index is calculated by combining the alkane degradation index, aromatic degradation index, and residual oil concentration to determine the bioremediation process and endpoint.

[0034] Furthermore, the oil spill degradation efficiency evaluation index in step five is calculated using the following formula: In the formula, ODEI is the oil spill degradation efficiency evaluation index; ADI is the alkane degradation index, with a value range of [value missing]. ArDI is the aromatics degradation index, with a value range of... ; This refers to the residual oil concentration. Initial oil concentration; , , Let be the weighting coefficient, satisfying Preferred , , .

[0035] Furthermore, the detection sensitivity of the kit is as follows: detection limit for alkane degradation intermediates. Fatty alcohol equivalents; detection limits for aromatic degradation intermediates Catechol equivalent; Limit of detection for total petroleum hydrocarbon residues .

[0036] Furthermore, the reagent kit in Under conditions of protection from light, the shelf life is not less than [number] years. Months.

[0037] The beneficial effects of this invention are:

[0038] The in-situ detection kit for oil spill degradation intermediates provided by this invention has the following significant advantages:

[0039] First, it enables rapid on-site detection of oil spill degradation. This invention employs a dual-signal response mechanism of enzyme-catalyzed colorimetry and fluorescence quenching, reducing the time required from sample processing to obtaining detection results to only a short time. Minutes, compared to traditional gas chromatography-mass spectrometry analysis methods The testing cycle is reduced, and efficiency is improved by more than [percentage missing]. This doubles the performance, meeting the needs of real-time monitoring at the bioremediation site.

[0040] Second, this invention achieves, for the first time, the specific detection of intermediate products of petroleum hydrocarbon degradation. The three-module system designed in this invention targets fatty alcohols (alkane degradation products), catechol compounds (aromatic degradation products), and total petroleum hydrocarbon residues, respectively, and can distinguish the degradation contribution of different types of petroleum hydrocarbons, a function that traditional detection methods cannot achieve.

[0041] Third, the detection cost is significantly reduced. The cost of a single test using the kit of this invention is approximately [missing information]. Yuan, compared to gas chromatography-mass spectrometry analysis The cost per unit was reduced. That's all. The price of a portable photometer is approximately... Yuan, far lower than that of gas chromatography-mass spectrometry. An investment of 10,000 yuan.

[0042] Fourth, it is easy to operate and requires no professional technicians. The detection operation of this invention only includes three simple steps: sample filtration, reagent addition, and instrument reading. These steps can be completed independently by field staff after simple training, thus lowering the technical threshold.

[0043] Fifth, the synergistic use of colorimetric and fluorescence signals improves detection reliability. The alkane degradation indicator module and the aromatics degradation indicator module employ enzymatic colorimetry, suitable for semi-quantitative rapid screening of colored samples; the total petroleum hydrocarbon residue detection module uses fluorescence quenching, offering higher sensitivity and suitable for precise quantification of low-concentration residual oil. The complementary nature of these two signal modes enhances the reliability of the detection results.

[0044] Sixth, this invention provides a standardized evaluation tool for oil spill bioremediation projects. The oil spill degradation efficiency evaluation index established in this invention comprehensively considers three dimensions: alkane degradation, aromatic degradation, and residual oil concentration, providing a scientific basis for the quantitative assessment of bioremediation effects and the determination of project endpoints. Attached Figure Description

[0045] Figure 1 This is a standard curve of the alkane degradation index.

[0046] Figure 2 This is a standard curve of the aromatic hydrocarbon degradation index.

[0047] Figure 3 This is a graph showing the linear relationship between residual oil concentration and fluorescence quenching percentage. Detailed Implementation

[0048] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are only for illustrating the present invention and are not intended to limit the scope of protection of the present invention.

[0049] Example 1: Preparation of an alkane degradation indicator module

[0050] This embodiment provides a detailed preparation method for an alkane degradation indicator module.

[0051] Preparation of lyophilized fatty alcohol dehydrogenase powder: The long-chain fatty alcohol dehydrogenase gene from *Bacillus stearothermophilus* was cloned into the pET-28a expression vector after codon optimization, and then transformed into *E. coli* BL21(DE3) competent cells. Cultured under the conditions until achieve Add the final concentration. Isopropyl-β-D-thiogalactoside induces expression. Continue to cultivate Hours. Collect bacterial cells, sonicate to disrupt, centrifuge to collect the supernatant, purify by nickel affinity chromatography, and collect the target protein. Dialyze the purified protein to a concentration of [missing information - likely a specific concentration]. Trehalose and The enzyme was prepared by dispensing and freeze-drying in a polyvinylpyrrolidone protective agent solution to obtain an enzyme with a specific activity. Lyophilized powder of fatty alcohol dehydrogenase.

[0052] Coenzyme Preparation of stock solution: Weigh out Oxidized nicotinamide adenine dinucleotide soluble in Prepared in Tris-HCl buffer (pH 8.0) of Stock solution. Dispensed into... In centrifuge tubes, save.

[0053] Preparation of tetrazolium salt colorimetric solution: Weigh out... Iodonitrazole purple dissolves in Add to deionized water Phenyrazine methyl sulfate, as an electron transfer medium, should be stored away from light and formulated as follows: Tetrazolium salt colorimetric solution.

[0054] Preparation of carbon chain length selective regulator: Weigh out Tween-80 dissolves in In phosphate buffer, it is prepared as follows: The regulator solution. This regulator selectively promotes [the process] by forming micellar structures. - The binding of medium- and long-chain fatty alcohols to enzymes.

[0055] Assembly of the alkane degradation indicator module: Fatty alcohol dehydrogenase lyophilized powder pre-filled in The bottom of the transparent polypropylene centrifuge tube is sealed and then protected with nitrogen gas. (Included with accessories) Stock solution, Tetrazazole salt colorimetric solution and Carbon chain length selective modifiers, individually packaged.

[0056] Performance verification: formulation , , , , , , The n-dodecyl alcohol standard solution was analyzed according to the kit instructions. Take... The standard solution was added to a detection tube pre-filled with fatty alcohol dehydrogenase lyophilized powder, and then... Carbon chain length selective regulators Stock solution and Tetrazazole salt colorimetric solution reaction Minutes later, using a portable photometer The absorbance was measured at [location]. For example... Figure 1As shown, absorbance is related to n-dodecyl alcohol concentration. The relationship exhibits good linearity within the range, and the linear equation is: Correlation coefficient The detection limit is .

[0057] Example 2: Preparation of an aromatic hydrocarbon degradation indicator module

[0058] This embodiment provides a detailed preparation method for an aromatic hydrocarbon degradation indicator module.

[0059] Preparation of lyophilized catechol 2,3-dioxygenase powder: The catechol 2,3-dioxygenase gene from *Pseudomonas putida* was amplified by PCR and cloned into the pET-32a expression vector. This was then transformed into *Escherichia coli* BL21(DE3) and cultured in LB medium. Cultured to the logarithmic growth phase, then added Isopropyl-β-D-thiogalactoside induction, with the addition of Ferrous ammonium sulfate serves as an iron source. Induced expression Hours. After ultrasonic disruption, the supernatant was purified sequentially by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration chromatography. The purified enzyme protein was then... sucrose, Mannitol and Reduced glutathione was mixed and freeze-dried to obtain an enzyme with an activity of [value missing]. Lyophilized powder of catechol dioxygenase.

[0060] Preparation of activating solution: Weigh out Ferrous ammonium sulfate and Ascorbic acid soluble in Deionized water is prepared to contain and Ascorbic acid activation solution. Ascorbic acid acts as a reducing agent to prevent... Oxidized to Prepare and use immediately or Store frozen.

[0061] Preparation of reaction buffer: Potassium dihydrogen phosphate and Dipotassium hydrogen phosphate dissolves in Adjust the pH of the deionized water to... Formulated Phosphate buffer solution.

[0062] Preparation of aromatic ring number selectivity modifier: Weigh out Hydroxypropyl-β-cyclodextrin is soluble in Prepared in reaction buffer solution The regulating agent solution. Hydroxypropyl-β-cyclodextrin selectively solubilizes by forming inclusion complexes. Cyclic catechol substrates improve detection sensitivity.

[0063] Assembly of the aromatics degradation indicator module: Catechol dioxygenase lyophilized powder pre-filled in Brown, light-proof centrifuge tubes with nitrogen-filled, sealed bottoms. (Included with accessories) Activation solution, reaction buffer and Aromatic ring number selectivity modifiers are individually packaged.

[0064] Performance verification: formulation , , , , , , The catechol standard solution was tested according to the kit instructions. Take... The standard solution was added to a detection tube pre-filled with catechol dioxygenase lyophilized powder, and then... Aromatic ring number selectivity modifiers Activation solution and reaction buffer, reaction Minutes later, using a portable photometer The absorbance was measured at [location]. For example... Figure 2 As shown, absorbance is related to catechol concentration. The relationship exhibits good linearity within the range, and the linear equation is: Correlation coefficient The detection limit is .

[0065] Meanwhile, other catechol compounds such as 4-methylcatechol and 3,4-dihydroxybenzoic acid were also detected. The results showed that the module had a good response to a variety of aromatic hydrocarbon degradation intermediates, demonstrating broad-spectrum substrate adaptability.

[0066] Example 3: Preparation of a total petroleum hydrocarbon residue detection module

[0067] This embodiment provides a detailed preparation method for a total petroleum hydrocarbon residue detection module.

[0068] Synthesis of fluorescein-labeled cyclodextrin probes: Take Hydroxypropyl-β-cyclodextrin is soluble in Add to anhydrous dimethyl sulfoxide Carbodiimide and Hydroxysuccinimide activates the hydroxyl group of hydroxypropyl-β-cyclodextrin. Reaction Hours later, add fluorescein isothiocyanate (fluorescein to cyclodextrin molar ratio) ), room temperature, protected from light, reaction Hours. The reaction solution was dialyzed (molecular weight cutoff). After removing unreacted small molecules, freeze-drying yields a modified product with approximately [percentage missing]. The crude probes, consisting of one fluorescein molecule and one cyclodextrin molecule, were further purified by preparative high-performance liquid chromatography (HPLC). The target peaks were collected and freeze-dried to obtain high-purity fluorescein-labeled cyclodextrin probes.

[0069] Preparation of probe solution: Weigh out Fluorescein-labeled cyclodextrin probe dissolved in In phosphate buffer (pH 7.4), it is prepared as follows: The probe solution.

[0070] Preparation of fluorescence stabilizer: Weigh out Sodium ascorbate, Disodium ethylenediaminetetraacetate and Tween-20 dissolves in In phosphate buffer, a fluorescence stabilizer is prepared. Sodium ascorbate and disodium EDTA act as antioxidants and chelates metal ions, while Tween-20 is used as a solubilizer.

[0071] Preparation of hydrophobicity enhancer: Weigh out Sodium deoxycholate dissolves in In phosphate buffer, it is prepared as follows: A hydrophobicity enhancer. Sodium deoxycholate, after forming a complex with cyclodextrin, can enhance the binding ability of its hydrophobic cavities to petroleum hydrocarbons.

[0072] Assembly of the total petroleum hydrocarbon residue detection module: probe solution, Hydrophobic enhancers and Fluorescent stabilizers were premixed and dispensed into In brown, light-proof centrifuge tubes, Store away from light.

[0073] Performance verification: Using No. 0 diesel as a model petroleum hydrocarbon, a formulation was prepared. , , , , , , , The standard solution. Take The standard solution was added to the detection tube containing the pre-packed probe mixture and vortexed to mix. Minutes later, using a portable photometer excitation, Fluorescence intensity was measured under emission conditions. The fluorescence intensity of the blank sample was used as the baseline. The fluorescence intensity of the sample is Calculate the percentage of fluorescence quenching. .

[0074] like Figure 3 As shown, the percentage of fluorescence quenching is related to the diesel concentration. The relationship exhibits good linearity within the range, and the linear equation is: Correlation coefficient The detection limit is When the diesel concentration is At that time, the percentage of fluorescence quenching reached This indicates that the probe has a wide dynamic detection range.

[0075] Further testing was conducted on the probe's response to different types of petroleum hydrocarbons, including crude oil, gasoline, and lubricating oil. The results showed that the probe responded well to all types of petroleum hydrocarbons, and the order of fluorescence quenching efficiency was: crude oil > diesel > lubricating oil > gasoline. This is related to the differences in the content of polycyclic aromatic hydrocarbons and long-chain alkanes in petroleum hydrocarbons.

[0076] Example 4: Design of a Portable Photometer

[0077] This embodiment provides a design scheme for a portable photometer.

[0078] Optical system design: The photometer adopts a dual-optical-path design and is equipped with... , , Three wavelengths of light-emitting diodes are used as light sources, and An ultraviolet light-emitting diode (UVLED) is used as the fluorescence excitation source. The detector employs a silicon photodiode, in conjunction with... Narrow-band filters are used for fluorescence detection. The sample cell is standard. Optical path cuvette or dedicated Detection tube adapter.

[0079] Electronic system design: A low-power microprocessor (STM32L476) is used for data acquisition and processing. Integration. The bit-level analog-to-digital converter ensures detection accuracy and sampling frequency. .built-in , Lithium battery, can work continuously when fully charged. More than 24 hours. Configuration inch color LCD screen and The function buttons are used for human-computer interaction.

[0080] Software system design: The built-in data processing software contains pre-stored standard curves for alkane degradation index, aromatic degradation index, and residual oil concentration. After the user selects a detection mode, the software automatically calls up the corresponding wavelength light source and detection parameters. After the detection is completed, the software automatically calculates the concentration value based on the measured absorbance or fluorescence intensity, and further calculates the oil spill degradation efficiency evaluation index. Data can be exported to a smartphone or computer via Bluetooth or USB interface.

[0081] Technical Specifications: Absorbance Measurement Range Precision Fluorescence detection sensitivity Rhodamine B equivalent; dimensions ;weight Protection rating: IP54.

[0082] Example 5: Application Validation of the Reagent Kit - Simulated Oil Spill Biodegradation Experiment

[0083] This embodiment verifies the practical application effect of the kit through a simulated oil spill biodegradation experiment.

[0084] Experimental setup: Prepare a mixture containing... Artificial seawater culture medium for No. 0 diesel was inoculated with enriched bacterial cultures from the polluted sea area of ​​the Dalian New Port oil spill, and the bacterial concentration was... .set up Group of parallel experimental groups and Group aseptic control group , Shaking culture under certain conditions Day. On the respective days. , , , , , Samples were taken daily and detected in parallel using the kit of this invention and the gas chromatography-mass spectrometry method.

[0085] Kit test results:

[0086] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0087] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0088] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0089] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0090] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0091] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index .

[0092] Gas chromatography-mass spectrometry analysis results: (Number) The right time, n-alkanes ( - The total degradation rate is The total degradation rate of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene, pyrene, etc.) is Total petroleum hydrocarbon degradation rate .

[0093] Results Analysis: The alkane degradation index and aromatic degradation index measured by the kit showed good correlation with the degradation rates of n-alkane and polycyclic aromatic hydrocarbons analyzed by gas chromatography-mass spectrometry (correlation coefficients were respectively...). and The relative deviation between the residual oil concentration measured by the kit and the total petroleum hydrocarbon residues analyzed by gas chromatography-mass spectrometry is less than [value missing]. The above results demonstrate that the kit of the present invention can accurately reflect the process of oil spill biodegradation.

[0094] Repeatability verification: for the first Samples were taken on the day. After two parallel tests, the relative standard deviation of the alkane degradation index was [value missing]. The relative standard deviation of the aromatic degradation index is The relative standard deviation of residual oil concentration is This indicates that the kit has good reproducibility.

[0095] Example 6: Application Validation of the Reagent Kit – Actual Oil Spill Remediation Site

[0096] This example demonstrates the application effect of the reagent kit in a real oil spill bioremediation site.

[0097] Application scenario: An oil spill occurs in a certain sea area, with a spill volume of approximately... Tons of diesel fuel. An emergency response was implemented using a bioremediation approach involving microbial agent spraying and nutrient-enhanced treatment. [The following appears to be unrelated and possibly a separate sentence fragment:] ...set up in the remediation area... The monitoring points were located on the [number]th day after the start of bioremediation. , , , , , On-site monitoring will be conducted daily.

[0098] Monitoring results: Taking monitoring point No. as an example, the changes of each detection indicator over time are as follows:

[0099] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0100] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0101] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0102] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0103] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0104] No. Day: Alkane Degradation Index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index .

[0105] The average oil spill degradation efficiency evaluation index of the monitoring points was in the [number]th [period]. Tianda This indicates that the bioremediation effect is good. According to the test results from the kit, on the [date / time]... The evaluation index of oil spill degradation efficiency exceeded [percentage] within approximately [number] days. The threshold indicates that the enhanced repair measures can be terminated.

[0106] Comparison with laboratory methods: A portion of the sample was sent to the laboratory for gas chromatography-mass spectrometry analysis. The results showed that the relative deviation between the residual oil concentration measured by the kit and the laboratory analysis value was within [value missing]. Within the specified range, it meets the accuracy requirements for rapid on-site screening.

[0107] On-site operation experience: The entire testing process for each sample takes approximately... Minutes, including sample pretreatment Minutes, three-module detection Minutes. Single person can Complete within hours The complete testing of all locations significantly improved the efficiency of on-site monitoring.

[0108] Example 7: Comparison of detection performance for different types of petroleum hydrocarbons

[0109] This example compares the detection performance of the kit for different types of petroleum hydrocarbons.

[0110] Sample preparation: Prepare samples containing... crude, diesel fuel, gasoline and Artificial seawater samples of lubricating oil were inoculated with petroleum hydrocarbon-degrading bacteria. nourish sky.

[0111] Test results:

[0112] Crude oil sample: Alkane degradation index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0113] Diesel sample: Alkane degradation index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0114] Gasoline sample: Alkane degradation index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index ;

[0115] Lubricating oil sample: Alkane degradation index Aromatic hydrocarbon degradation index residual oil concentration Oil spill degradation efficiency evaluation index .

[0116] Results Analysis: Significant differences were observed in the degradation characteristics of different types of petroleum hydrocarbons. Diesel and crude oil exhibited high alkane degradation indices, reflecting the ease with which their n-alkane components are degraded by microorganisms. Gasoline showed a high aromatic hydrocarbon degradation index, consistent with its composition rich in monocyclic aromatic hydrocarbons (benzene, toluene, xylene). Lubricating oil showed the lowest degradation efficiency, likely due to its high content of antioxidants and high molecular weight components, which have a certain inhibitory effect on microorganisms. These results demonstrate that the kit of this invention can distinguish the degradation characteristics of different types of petroleum hydrocarbons, providing a basis for developing targeted remediation strategies.

[0117] Example 8: The Influence of Environmental Factors on Detection Performance

[0118] This example examines the effects of environmental factors such as temperature and salinity on the detection performance of the reagent kit.

[0119] Temperature effect experiment: In , , , , Under the same conditions, standard samples of the same concentration were tested respectively. The results showed that the three detection modules performed well. The test results were stable within the range, with a relative standard deviation of less than [value missing]. ; When enzyme activity decreases, the detection response decreases by approximately [percentage missing]. However, by extending the reaction time to Minutes can be compensated; Enzyme activity decreases significantly during this period, leading to reduced detection accuracy. Therefore, it is recommended to... Use the kit in the specified environment.

[0120] Experiment on the effect of salinity: In , , , , , Standard samples were tested under salinity conditions. Results showed that the alkane degradation indicator module and the aromatics degradation indicator module... The test results are stable within the salinity range; Enzyme activity decreased slightly under high salinity conditions. The total petroleum hydrocarbon residue detection module remained stable across the entire salinity range because the fluorescence quenching mechanism is independent of enzyme activity.

[0121] Interference Effect Experiment: Investigating the interference of common marine pollutants on detection, including heavy metal ions ( , , , ,concentration ), surfactant (sodium dodecyl sulfate, concentration) ) and humus (concentration) The results showed that... Concentration exceeding It has a slight inhibitory effect on the alkane degradation indicator module; the humic concentration exceeds There is a slight interference with the aromatics degradation indicator module. This interference can be eliminated through sample pretreatment steps (dilution or chelation).

[0122] Example 9: Stability study of the reagent kit

[0123] This embodiment examines the stability of the reagent kit under different storage conditions.

[0124] Storage conditions settings: Place the reagent kit in... , , The samples were stored at three different temperatures, and samples were taken periodically to test the activity and detection performance of each component.

[0125] Stability results:

[0126] Storage: Lyophilized powder of fatty alcohol dehydrogenase Enzyme activity retention rate after one month The above; catechol dioxygenase lyophilized powder Enzyme activity retention rate after one month The above; fluorescein-labeled cyclodextrin probe solution Fluorescence intensity retention rate after one month above.

[0127] Storage: Lyophilized powder of fatty alcohol dehydrogenase Enzyme activity retention rate after one month ; Catechol dioxygenase lyophilized powder Enzyme activity retention rate after one month ; Fluorescein-labeled cyclodextrin probe solution Fluorescence intensity retention rate after one month .

[0128] Storage: Lyophilized powder of fatty alcohol dehydrogenase After one month, the enzyme activity retention rate decreased to ; Catechol dioxygenase lyophilized powder After one month, the enzyme activity retention rate decreased to ; Fluorescein-labeled cyclodextrin probe solution Fluorescence intensity retention rate after one month .

[0129] Conclusion: The kit is effective in... Store in a dark place; shelf life is up to [date missing]. Months. In Storage under these conditions can further extend the shelf life. It is not recommended to store it under these conditions. Store at the above temperatures for extended periods.

[0130] Example 10: Comprehensive Mechanism Analysis

[0131] The three detection modules of the kit of this invention are based on different biochemical principles and work together to achieve a comprehensive evaluation of oil spill degradation.

[0132] The working mechanism of the alkane degradation indicator module: The first step in the microbial degradation of n-alkanes in petroleum hydrocarbons is terminal hydroxylation, generating the corresponding fatty alcohols. This invention utilizes fatty alcohol dehydrogenases to catalyze the oxidation of fatty alcohols to fatty aldehydes, while simultaneously providing coenzymes... It is reduced to NADH. NADH has the ability to reduce tetrazolium salts to formazan compounds, and the reaction requires the participation of the electron transfer medium methyl phenazine sulfate. The formazan compound is purple-red. A strong absorption peak is observed, and the absorbance is directly proportional to the concentration of fatty alcohol. This cascade enzymatic-chemical reaction achieves a highly efficient conversion from alkane degradation intermediates to detectable optical signals. Fatty alcohol dehydrogenase... - Fatty alcohols of varying carbon chain lengths exhibit good activity and can reflect the degradation contribution of alkanes with different carbon chain lengths.

[0133] The working mechanism of the aromatic hydrocarbon degradation indicator module: The microbial degradation pathway of polycyclic aromatic hydrocarbons (PAHs) includes two stages: upstream hydroxylation and downstream ring-opening. The upstream pathway converts PAHs into catechol compounds, while the downstream pathway involves ring-opening catalyzed by catechol dioxygenase. This invention selects to detect the downstream ring-opening product rather than the upstream hydroxylation product because catechol compounds are common intermediates in various PAH degradation pathways and are more representative. Catechol 2,3-dioxygenase catalyzes the reaction of catechol with molecular oxygen, in... - The aromatic ring is cleaved at different positions to form a yellow 2-hydroxymufoamic acid semialdehyde. This product... It exhibits a characteristic absorption peak. The active site of catechol dioxygenase contains... , The ascorbic acid in the activation solution plays a role in maintaining the reduced state of iron ions, ensuring the stable performance of enzyme activity.

[0134] Working Mechanism of the Total Petroleum Hydrocarbon Residue Detection Module: The fluorescein-labeled cyclodextrin probe designed in this invention is based on host-guest chemistry and the principle of fluorescence quenching. β-Cyclodextrin possesses a hydrophobic inner cavity and a hydrophilic outer surface, enabling it to form inclusion complexes with hydrophobic components in petroleum hydrocarbons. When petroleum hydrocarbon molecules enter the cyclodextrin cavity, they alter the microenvironment of the fluorescein, leading to fluorescence quenching. The quenching mechanism involves two processes: photoinduced electron transfer and energy transfer. Hydroxypropyl substitution improves the water solubility and inclusion capacity of cyclodextrin, while the addition of sodium deoxycholate further enhances the hydrophobicity of the cavity, improving the probe's binding efficiency to petroleum hydrocarbons. The degree of fluorescence quenching is positively correlated with the concentration of petroleum hydrocarbons, enabling the quantitative detection of residual oil concentration.

[0135] The overall evaluation logic of the three modules is as follows: the alkane degradation index reflects the degradation activity of n-alkane components, the aromatics degradation index reflects the degradation activity of polycyclic aromatic hydrocarbon components, and the residual oil concentration reflects the overall removal effect of petroleum hydrocarbons. These three indicators evaluate biodegradation efficiency from two dimensions: accumulation of metabolic intermediates and substrate consumption, respectively, mutually reinforcing each other and improving the reliability of the evaluation results. The oil spill degradation efficiency evaluation index integrates the three indicators, and the weighting coefficients can be adjusted according to the actual oil spill type and remediation target. When the oil spill is mainly composed of n-alkanes, the weight of the alkane degradation index can be appropriately increased; when the oil spill is rich in polycyclic aromatic hydrocarbons, the weights of the aromatics degradation index and residual oil concentration can be increased. This flexible evaluation system can adapt to the monitoring needs of different oil spill accidents.

[0136] Compared with existing technologies, the innovations of this invention are reflected in the following aspects: First, it proposes a strategy of detecting degradation intermediates rather than simply monitoring substrate consumption, which can more directly reflect the degree of microbial metabolic activity; second, it designs a dual-signal response system of colorimetry and fluorescence, taking into account the needs of qualitative screening and quantitative analysis; third, it integrates three functional modules into an integrated reagent kit, realizing the simultaneous and rapid detection of alkane degradation, aromatic degradation, and residual oil concentration; and fourth, it establishes an evaluation index for oil spill degradation efficiency, providing a standardized evaluation tool for bioremediation projects.

[0137] The above description is only a preferred embodiment of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. The method of using the in-situ detection kit for oil spill degradation intermediates, characterized in that, Includes the following steps: Sample collection and pretreatment steps: Samples were collected at the oil spill bioremediation site and then... Membrane filtration; Alkane degradation indicator module detection steps: Take the pretreated sample and add it to a detection tube pre-loaded with fatty alcohol dehydrogenase lyophilized powder, then add coenzyme. The stock solution and tetrazolium salt colorimetric solution were reacted, and the absorbance was measured afterward. For the aromatic hydrocarbon degradation indicator module detection step, the pretreated sample was added to a detection tube pre-loaded with catechol dioxygenase lyophilized powder, and then... The activation solution and reaction buffer were prepared, and the absorbance was measured after the reaction. In the total petroleum hydrocarbon residue detection step, the pretreated sample was added to a detection tube pre-loaded with a fluorescein-labeled cyclodextrin probe solution, and the percentage of fluorescence quenching was measured. In the comprehensive evaluation step, the oil spill degradation efficiency evaluation index was calculated based on the data processing software. The in-situ detection kit for oil spill degradation intermediates includes an alkane degradation indicator module, an aromatic hydrocarbon degradation indicator module, a total petroleum hydrocarbon residue detection module, and supporting portable photometer and data processing software; the alkane degradation indicator module contains lyophilized fatty alcohol dehydrogenase powder and coenzyme. The stock solution and tetrazolium salt colorimetric solution; the aromatic hydrocarbon degradation indicator module includes catechol dioxygenase lyophilized powder, The activation solution and reaction buffer; the total petroleum hydrocarbon residue detection module includes a fluorescein-labeled cyclodextrin probe solution and a fluorescence stabilizer.

2. The method of use according to claim 1, characterized in that, The lyophilized powder of fatty alcohol dehydrogenase was prepared by purifying and freeze-drying a long-chain fatty alcohol dehydrogenase expressed by recombinant Escherichia coli, with an enzyme activity of not less than [specified value]. The coenzyme The concentration of the stock solution is pH value The concentration of tetrazolium salt in the tetrazolium salt colorimetric solution is [missing information]. The tetrazolium salt is selected from one or more of 2,3,5-triphenyltetrazolium chloride, iodonitroxytetrazolium violet, or nitrobluetetrazolium.

3. The method of use according to claim 1, characterized in that, The lyophilized catechol dioxygenase powder was prepared by lyophilizing catechol 2,3-dioxygenase derived from recombinant Pseudomonas aeruginosa, and the enzyme activity was not less than [specified value]. The The activation solution contains Ferrous ammonium sulfate and Ascorbic acid; the reaction buffer solution has a pH value of Phosphate buffer or Tris-HCl buffer, concentration of .

4. The method of use according to claim 1, characterized in that, The fluorescein-labeled cyclodextrin probe is a β-cyclodextrin or hydroxypropyl-β-cyclodextrin covalently modified with fluorescein isothiocyanate, with a modification degree of [insert degree here]. Each cyclodextrin molecule contains one luciferin molecule, and the probe concentration is [missing value]. The fluorescence stabilizer contains antioxidants and The solubilizer.

5. The method of use according to claim 1, characterized in that, The alkane degradation indicator module further includes a carbon chain length selective regulator, which is selected from one or more combinations of polyethylene glycol monomethyl ether, Tween-80, or Span-20, at a concentration of [missing information]. .

6. The method of use according to claim 5, characterized in that, The aromatic hydrocarbon degradation indicator module further includes an aromatic ring number selectivity regulator, which is selected from one or more combinations of methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, or sulfobutyl ether-β-cyclodextrin, and has a concentration of [missing information]. .

7. The method of use according to claim 6, characterized in that, The fluorescein-labeled cyclodextrin probe in the total petroleum hydrocarbon residue detection module is further compounded with a hydrophobic enhancer. The hydrophobic enhancer is selected from one or more of sodium cholate, sodium deoxycholate, or sodium chenodeoxycholate, and the compounding ratio is the molar ratio of cyclodextrin to the hydrophobic enhancer. to .

8. The method of use according to claim 1, characterized in that, In the detection step of the alkane degradation indicator module, reaction under conditions ,exist The absorbance was measured at the specified location; in the detection step of the aromatic hydrocarbon degradation indicator module, the absorbance was measured at the specified location. reaction under conditions ,exist The absorbance was measured at the specified location; in the total petroleum hydrocarbon residue detection step, at... excitation, Fluorescence intensity was measured under emission conditions.

9. The method of use according to claim 1, characterized in that, The oil spill degradation efficiency evaluation index in the comprehensive evaluation step is calculated using the following formula: In the formula, ODEI is the oil spill degradation efficiency evaluation index, ADI is the alkane degradation index, and ArDI is the aromatics degradation index. This refers to the residual oil concentration. This represents the initial oil concentration. , , For the weighting coefficients, satisfying .

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