Method for detecting biochemical oxygen demand in sewage

Abstract

The application provides a method for detecting biochemical oxygen demand in sewage, and belongs to the field of oxygen demand detection. The method comprises the following steps: forming a fluorine-containing organosilicon nano coating on the electrode surface of a dissolved oxygen sensor of a full-automatic BOD5 analyzer to obtain a fluorine-silicon modified dissolved oxygen sensor; pretreating the sewage to be detected in an adsorption column composed of modified activated carbon and porous silica gel to obtain a pretreated water sample; fixing bacillus subtilis on a porous carrier and then putting the carrier into the pretreated water sample to obtain an inoculated water sample; measuring the initial dissolved oxygen of the inoculated water sample and the end-point dissolved oxygen after 5-day culture; and obtaining the five-day biochemical oxygen demand in the sewage according to the initial dissolved oxygen and the end-point dissolved oxygen. Through the four-level collaborative system of "sensor anti-interference optimization, sample purification and enrichment, microbial reaction standardization and accurate signal acquisition", the application systematically eliminates the error sources in the traditional BOD5 detection, and realizes the accurate measurement of the organic matter pollution level in complex sewage.

Description

Technical Field

[0001] This application relates to the field of oxygen demand detection technology, and in particular to a method for detecting biochemical oxygen demand in wastewater. Background Technology

[0002] Biochemical oxygen demand (BOD), as an important indicator reflecting the degree of organic pollution in water bodies, is widely used in water quality monitoring, environmental assessment, and wastewater treatment process optimization. Traditional five-day BOD5 measurement methods, due to their long experimental cycle (generally 6-7 days), numerous experimental steps, and reliance on manual operation, are susceptible to various interference factors during the experiment, making it difficult to effectively guarantee the accuracy and repeatability of the measurement results, thus affecting the scientific validity and reliability of water quality assessment.

[0003] In recent years, with the continuous development of automated instrument technology, fully automated BOD5 analyzers have begun to be widely used in environmental monitoring and other fields. These fully automated instruments employ advanced sensor technology and computer control systems, enabling real-time monitoring of key parameters such as temperature, humidity, and dissolved oxygen electrodes, thereby significantly improving the accuracy and repeatability of the detection data. Through automated detection processes, fully automated BOD5 analyzers not only overcome the errors of manual operation in traditional methods but also significantly improve detection efficiency and reduce the impact of external interference on experimental results. However, dissolved oxygen sensors are susceptible to contamination by biofilms (such as algae and bacteria) and chemical residues in water bodies, leading to drift or failure of detection values, requiring frequent cleaning or replacement. Furthermore, inhibitory substances such as heavy metal ions, macromolecular humic acid, and residual chlorine in wastewater are difficult to completely remove, resulting in reduced microbial activity and deviations in BOD detection values. Therefore, improving the detection accuracy of five-day biochemical oxygen demand in wastewater based on fully automated BOD5 analyzers is a pressing technical problem that needs to be solved. Summary of the Invention

[0004] This application provides a method for detecting biochemical oxygen demand (BOD) in wastewater to address the following technical problem: how to improve the detection accuracy of five-day BOD in wastewater based on a fully automated BOD5 analyzer.

[0005] This application provides a method for detecting biochemical oxygen demand (BOD) in wastewater, the method comprising:

[0006] A fluorinated organosilicon nano-coating was formed on the electrode surface of the dissolved oxygen sensor in a fully automated BOD5 analyzer to obtain a fluorinated silicon modified dissolved oxygen sensor.

[0007] The wastewater to be tested was pretreated in an adsorption column composed of modified activated carbon and porous silica gel to remove inhibitory substances and enrich degradable organic matter, thus obtaining a pretreated water sample.

[0008] Bacillus subtilis was immobilized on a porous carrier and then added to the pretreated water sample to obtain an inoculated water sample.

[0009] The initial dissolved oxygen and the endpoint dissolved oxygen after 5 days of incubation were determined using the fully automated BOD5 analyzer.

[0010] The five-day biochemical oxygen demand (BOD) in wastewater is obtained based on the initial dissolved oxygen and the endpoint dissolved oxygen.

[0011] Optionally, the thickness of the fluorinated organosilicon nanocoating is 50–100 nm, and the contact angle is ≥110°.

[0012] Optionally, the preparation method of the fluorinated organosilicon nanocoating includes:

[0013] γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate and acetic acid were added to an ethanol / water mixed solvent to carry out a hydrolysis-condensation reaction to obtain the first sol;

[0014] Nano-SiO2 particles were added to the first sol and dispersed by ultrasonication to obtain the second sol.

[0015] Methyl methacrylate and ammonium persulfate are added to the second sol to carry out a copolymerization reaction to obtain a coating.

[0016] The coating is applied to the electrode surface of the dissolved oxygen sensor and then thermally cured to form a fluorinated organosilicon nanocoating.

[0017] Optionally, the parameters of the hydrolysis-condensation reaction include: a reaction temperature of 60–80°C, a reaction time of 2–4 h, and a pH value adjusted to 4.0–6.0 by acetic acid;

[0018] The nano-SiO2 particles have a diameter of 10–50 nm and are added at a rate of 5–15% of the total mass of the first sol.

[0019] The power of ultrasonic dispersion is 200-400W, and the time is 30-60min;

[0020] The molar ratio of methyl methacrylate to N-methylperfluorooctyl sulfonyl amino acrylate is 2:1 to 4:1;

[0021] The amount of ammonium persulfate added is 0.5% to 2.0% of the total mass of the second sol;

[0022] The copolymerization reaction temperature is 70–80℃, and the reaction time is 2–3 hours.

[0023] The temperature for heat curing is 120–150℃, and the time is 1–2 hours.

[0024] Optionally, the mass ratio of the modified activated carbon to the porous silica gel is 3:1, and the dynamic adsorption flow rate of the adsorption column is 1 to 3 mL / min.

[0025] Optionally, the modified activated carbon is prepared by KOH activation and has a specific surface area >2000 m². 2 / g, for Hg 2+ The adsorption capacity is ≥200mg / g, and the removal rate of humic acid is ≥95%.

[0026] Optionally, the immobilization of Bacillus subtilis on a porous carrier includes:

[0027] Diatomaceous earth or activated carbon carriers were impregnated in Bacillus subtilis bacterial solution. The pH of the system was adjusted to 7.0–7.5 using phosphate buffer and kept at a constant temperature with shaking for 1–2 hours to ensure that the bacteria were fully adsorbed into the carrier pores.

[0028] Optionally, the loading concentration of Bacillus subtilis is 0.2 × 10⁻⁶. 7 ~19.7×10 7 CFU / mL;

[0029] The porosity of the diatomaceous earth or activated carbon is ≥90%.

[0030] Optionally, the volume ratio of the immobilized bacterial agent formed by Bacillus subtilis fixed on a porous carrier to the pretreated water sample is 0.1:1 to 0.2:1.

[0031] Optionally, the pretreated water sample is adjusted through the following steps:

[0032] pH adjustment: Adjust the pH of the water sample to 6.5–7.5 using phosphate buffer solution;

[0033] Dilution gradient: Based on the initial COD value of the water sample, no dilution is required when COD ≤ 100 mg / L, and when COD is 100–4000 mg / L, dilution is performed in a gradient of 1:5 to 1:10.

[0034] Sodium thiosulfate addition: Add sodium thiosulfate to the water sample containing residual chlorine to a final concentration of 10 mg / L, and let stand for 10 min to neutralize interference.

[0035] The technical solutions provided in this application have the following advantages compared with the prior art:

[0036] This application provides a method for detecting biochemical oxygen demand in wastewater.

[0037] Dissolved oxygen sensors are susceptible to interference from organic residues and biofilms, leading to decreased sensitivity. Firstly, this application employs a fluorinated organosilicon nanocoating on the electrode surface to improve detection accuracy through the following mechanisms: (1) Anti-fouling and anti-biofilm adhesion: Fluorinated groups have low surface energy, reducing the adsorption and deposition of organic matter and microorganisms on the electrode surface, thus preventing sensor signal drift; the organosilicon nanostructure forms a uniform protective layer, reducing direct contact between the electrode and pollutants, and extending the sensor's lifespan. (2) Improved oxygen mass transfer efficiency: The porous structure of the nanocoating allows oxygen to pass through quickly while blocking large molecular impurities, ensuring the real-time accuracy of dissolved oxygen (DO) measurement and avoiding DO measurement deviations caused by electrode contamination;

[0038] Secondly, this application optimizes water sample conditions through a dual adsorption-enrichment mechanism of modified activated carbon-porous silica gel adsorption column pretreatment: (1) Removal of inhibitory substances: Modified activated carbon adsorbs heavy metal ions and toxic organic substances (such as phenols and aldehydes) through surface functional groups (such as hydroxyl and carboxyl groups), and porous silica gel further adsorbs small molecule inhibitors, eliminating their toxic effects on microbial metabolism. (2) Enrichment of degradable organic matter: The large specific surface area of ​​activated carbon and the porous structure of silica gel physically adsorb organic matter (such as carbohydrates and proteins) that can be decomposed by microorganisms in wastewater, increasing the concentration of target pollutants in the pretreated water sample, making subsequent biochemical reactions more significant, and making DO changes easier to detect;

[0039] Finally, this application improves the stability of microbial reactions through immobilization and inoculation techniques of Bacillus subtilis: (1) Maintaining microbial activity and uniformity: The porous carrier provides an immobilization microenvironment for Bacillus subtilis, avoiding cell dispersion and loss in water samples, while ensuring the mass transfer efficiency of oxygen and organic matter, so as to make the microbial metabolic rate consistent. (2) Standardized reaction system: The inoculation amount of immobilized microorganisms is controllable, avoiding the difference in DO consumption caused by microbial concentration fluctuations in traditional inoculation methods, and improving the repeatability of detection;

[0040] This improves the accuracy of five-day biochemical oxygen demand detection in wastewater based on a fully automated BOD5 analyzer. Attached Figure Description

[0041] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0042] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 This is a schematic flowchart of a method for detecting biochemical oxygen demand in wastewater, provided as an embodiment of this application. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0045] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0046] Figure 1 This is a schematic flowchart of a method for detecting biochemical oxygen demand in wastewater, provided as an embodiment of this application.

[0047] like Figure 1 As shown, this application provides a method for detecting biochemical oxygen demand (BOD) in wastewater, the method comprising:

[0048] S1. A fluorinated organosilicon nano-coating is formed on the electrode surface of the dissolved oxygen sensor of a fully automated BOD5 analyzer to obtain a fluorinated silicon modified dissolved oxygen sensor.

[0049] It should be noted that the fully automated BOD5 (five-day biochemical oxygen demand) analyzer is a precision analytical instrument that automatically detects the degree of organic pollution in water samples such as sewage and surface water based on the principle that dissolved oxygen (DO) is consumed during the degradation of organic matter by microorganisms. Its core function is to automatically calculate the BOD5 value by monitoring DO changes in real time, replacing the traditional manual dilution and inoculation method, and significantly improving detection efficiency and accuracy.

[0050] Dissolved oxygen sensors are susceptible to interference from organic residues and biofilms, leading to decreased sensitivity. This application employs a fluorinated organosilicon nanocoating on the electrode surface to improve detection accuracy through the following mechanisms: (1) Anti-fouling and anti-biofilm adhesion: Fluorinated groups have low surface energy, which can reduce the adsorption and deposition of organic matter and microorganisms in wastewater on the electrode surface, avoiding sensor signal drift; the organosilicon nanostructure forms a uniform protective layer, reducing direct contact between the electrode and pollutants, and extending the sensor's service life. (2) Improved oxygen mass transfer efficiency: The porous structure of the nanocoating allows oxygen to pass through quickly while blocking large molecular impurities, ensuring the real-time and accurate measurement of dissolved oxygen (DO), and avoiding DO measurement deviations caused by electrode contamination. (3) Contribution to accuracy: Traditional sensors are susceptible to the adhesion of suspended solids and microorganisms in wastewater, leading to DO measurement errors; the modified coating can maintain the cleanliness of the electrode surface, making the measured values ​​of initial dissolved oxygen (DO0) and endpoint dissolved oxygen (DO5) closer to the true values.

[0051] In some embodiments, the thickness of the fluorinated organosilicon nanocoating is 50–100 nm, and the contact angle is ≥110°.

[0052] Limiting the coating thickness to 50–100 nm ensures complete coverage of the electrode surface, forming a continuous protective layer to prevent contaminant penetration; it also avoids excessive thickness that could increase oxygen mass transfer resistance and affect sensor response speed. A contact angle of ≥110° ensures the superhydrophobic surface reduces the adhesion of organic matter and microorganisms in wastewater, lowers the probability of biofilm formation, and extends sensor lifespan.

[0053] In some embodiments, the method for preparing the fluorinated organosilicon nanocoating includes:

[0054] γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate and acetic acid were added to an ethanol / water mixed solvent to carry out a hydrolysis-condensation reaction to obtain the first sol;

[0055] Nano-SiO2 particles were added to the first sol and dispersed by ultrasonication to obtain the second sol.

[0056] Methyl methacrylate and ammonium persulfate are added to the second sol to carry out a copolymerization reaction to obtain a coating.

[0057] The coating is applied to the electrode surface of the dissolved oxygen sensor and then thermally cured to form a fluorinated organosilicon nanocoating.

[0058] In some embodiments, the parameters of the hydrolysis-condensation reaction include: a reaction temperature of 60–80°C, a reaction time of 2–4 h, and a pH value adjusted to 4.0–6.0 by acetic acid.

[0059] The nano-SiO2 particles have a diameter of 10–50 nm and are added at a rate of 5–15% of the total mass of the first sol.

[0060] The power of ultrasonic dispersion is 200-400W, and the time is 30-60min;

[0061] The molar ratio of methyl methacrylate to N-methylperfluorooctyl sulfonyl amino acrylate is 2:1 to 4:1;

[0062] The amount of ammonium persulfate added is 0.5% to 2.0% of the total mass of the second sol;

[0063] The copolymerization reaction temperature is 70–80℃, and the reaction time is 2–3 hours.

[0064] The temperature for heat curing is 120–150℃, and the time is 1–2 hours.

[0065] Limiting the temperature (60–80℃) and time (2–4 h) of the hydrolysis-condensation reaction can promote the hydrolysis of silane monomers (γ-methacryloyloxypropyltrimethoxysilane) to generate silanol groups (Si-OH), which then condense to form a Si-O-Si network structure. Too low a temperature or too short a time will lead to incomplete reaction and poor coating adhesion. Limiting the pH value (4.0–6.0, adjusted with acetic acid) ensures a moderate silane hydrolysis rate under acidic conditions, preventing excessively rapid hydrolysis that could lead to particle agglomeration and ensuring coating uniformity.

[0066] Limit the addition of nano-SiO2 (particle size 10-50nm, addition amount 5-15%). Above 10nm, avoid agglomeration, below 50nm, ensure nanoscale roughness, and enhance the hydrophobicity and anti-fouling ability of the coating. The addition amount of more than 5% provides sufficient roughness, and less than 15% prevents the coating from becoming brittle and maintains flexibility.

[0067] Limited ultrasonic dispersion (200-400W, 30-60min) breaks down SiO2 agglomerates with high-intensity ultrasonication, making them uniformly dispersed in the sol and forming a nanoscale protrusion structure, which improves the surface roughness and hydrophobicity of the coating.

[0068] By limiting the copolymerization reaction parameters, the molar ratio of methyl methacrylate (MMA) to fluorine monomer is (2:1 to 4:1). MMA provides coating flexibility and adhesion, while the fluorine monomer (N-methylperfluorooctyl sulfonyl amino acrylate) imparts low surface energy. When the molar ratio is >4:1, insufficient fluorine content leads to a decrease in hydrophobicity; when it is <2:1, the coating brittleness increases.

[0069] The amount of ammonium persulfate (APS) added is limited (0.5% to 2.0%), which can act as an initiator to start the free radical polymerization reaction. Too little addition will lead to incomplete polymerization, while too much will generate short-chain polymers and reduce the coating strength.

[0070] Limiting the thermosetting conditions (120-150℃, 1-2h) can promote further condensation of residual silanol groups, enhancing the crosslinking density and adhesion of the coating; if the temperature is too low or the time is too short, the curing will be incomplete, resulting in poor corrosion resistance of the coating.

[0071] S2. The wastewater to be tested is pretreated in an adsorption column composed of modified activated carbon and porous silica gel to remove inhibitory substances and enrich degradable organic matter, thus obtaining a pretreated water sample.

[0072] This application optimizes water sample conditions through a dual adsorption-enrichment mechanism of modified activated carbon-porous silica gel adsorption column pretreatment: (1) Removal of inhibitory substances: Modified activated carbon adsorbs heavy metal ions and toxic organic substances (such as phenols and aldehydes) through surface functional groups (such as hydroxyl and carboxyl groups), and porous silica gel further adsorbs small molecule inhibitors, eliminating their toxic effects on microbial metabolism. (2) Enrichment of degradable organic matter: The large specific surface area of ​​activated carbon and the porous structure of silica gel physically adsorb organic matter (such as carbohydrates and proteins) that can be decomposed by microorganisms in wastewater, increasing the concentration of target pollutants in the pretreated water sample, making subsequent biochemical reactions more significant, and DO changes easier to detect. (3) Contribution to accuracy: If inhibitors are present in wastewater, it will lead to a decrease in microbial activity and a reduction in DO consumption, resulting in a lower BOD5 detection result; after pretreatment, interference can be eliminated, ensuring that the degradation reaction of organic matter by microorganisms proceeds normally.

[0073] Therefore, by filling the pretreatment column with modified activated carbon and porous silica gel, heavy metals (such as Hg) are dynamically adsorbed and removed. 2+ This process removes interfering macromolecules (such as humic acid) while retaining small organic molecules (such as acetic acid). Pretreatment removes interfering substances while preserving the target organic matter, providing optimized substrates for subsequent microbial degradation and reducing interference from non-target substances.

[0074] In some embodiments, the mass ratio of the modified activated carbon to the porous silica gel is 3:1, and the dynamic adsorption flow rate of the adsorption column is 1 to 3 mL / min.

[0075] The mass ratio of modified activated carbon to porous silica gel is 3:1. Activated carbon (3 parts) adsorbs organic matter and heavy metals through its large specific surface area, while silica gel (1 part) supplements the microporous structure to adsorb small molecule inhibitors. Too high a proportion of silica gel will reduce the overall adsorption capacity, while too low a proportion will affect the removal efficiency of small molecules. The adsorption flow rate is limited to 1-3 mL / min. ≤3 mL / min ensures sufficient contact between wastewater and adsorbent, while >1 mL / min avoids excessively long retention time that would lead to low treatment efficiency.

[0076] In some embodiments, the modified activated carbon is prepared by KOH activation and has a specific surface area >2000 m².2 / g, for Hg 2+ The adsorption capacity is ≥200mg / g, and the removal rate of humic acid is ≥95%.

[0077] KOH activation method: The carbon skeleton is etched by strong alkaline KOH to form a rich microporous structure with a specific surface area >2000m². 2 / g provides a high density of adsorption sites.

[0078] Limited Hg 2+ Adsorption capacity (≥200mg / g) and humic acid removal rate (≥95%): High mercury adsorption capacity ensures the removal of heavy metal inhibitors in wastewater, and humic acid removal prevents microorganisms from being encapsulated, ensuring the normal progress of subsequent biochemical reactions.

[0079] In some embodiments, the pretreated water sample is adjusted by the following steps:

[0080] pH adjustment: Adjust the pH of the water sample to 6.5–7.5 using phosphate buffer solution;

[0081] Dilution gradient: Based on the initial COD value of the water sample, no dilution is required when COD ≤ 100 mg / L, and when COD is 100–4000 mg / L, dilution is performed in a gradient of 1:5 to 1:10.

[0082] Sodium thiosulfate addition: Add sodium thiosulfate to the water sample containing residual chlorine to a final concentration of 10 mg / L, and let stand for 10 min to neutralize interference.

[0083] The optimal pH range for Bacillus subtilis is 6.5–7.5. Exceeding this range will inhibit enzyme activity (such as amylase and protease) and reduce the rate of organic matter decomposition.

[0084] Dilution gradient (based on COD value): No dilution is required for COD ≤ 100 mg / L, and low-concentration water samples are directly tested to avoid errors introduced by dilution; for COD ≤ 4000 mg / L, dilute at a ratio of 1:5 to 1:10: control the BOD5 value of the water sample within the linear range of instrument detection (usually 0.5 to 4000 mg / L), ensure that DO consumption does not exceed saturated dissolved oxygen (approximately 9 mg / L), and avoid measurement distortion.

[0085] Sodium thiosulfate removes chlorine (10 mg / L, let stand for 10 min). Residual chlorine (such as disinfection residue in tap water) has strong oxidizing properties, which can oxidize organic matter or inhibit microbial activity. 10 mg / L sodium thiosulfate can quantitatively reduce residual chlorine, and letting stand for 10 min ensures complete reaction.

[0086] S3. Immobilize Bacillus subtilis on a porous carrier and then add it to the pretreated water sample to obtain an inoculated water sample;

[0087] This application improves the stability of microbial reactions through immobilization and inoculation techniques using Bacillus subtilis: (1) Maintaining microbial activity and uniformity: The porous carrier provides an immobilized microenvironment for Bacillus subtilis, preventing cell dispersion and loss in water samples, while ensuring the mass transfer efficiency of oxygen and organic matter, thus ensuring a consistent metabolic rate of microorganisms. (2) Standardized reaction system: The inoculation amount of immobilized microorganisms is controllable, avoiding the DO consumption differences caused by microbial concentration fluctuations in traditional inoculation methods, and improving the repeatability of detection. (3) Contribution to accuracy: In traditional BOD5 detection, microbial activity is greatly affected by the inoculation source, which easily leads to discrete results; after immobilization, the amount and activity of microorganisms in each test can be ensured to be stable, making DO5 measurements more comparable.

[0088] In some embodiments, immobilizing Bacillus subtilis on a porous carrier includes:

[0089] Diatomaceous earth or activated carbon carriers were impregnated in Bacillus subtilis bacterial solution. The pH of the system was adjusted to 7.0–7.5 using phosphate buffer and kept at a constant temperature with shaking for 1–2 hours to ensure that the bacteria were fully adsorbed into the carrier pores.

[0090] In some embodiments, the loading concentration of Bacillus subtilis is 0.2 × 10⁻⁶. 7 ~19.7×10 7 CFU / mL;

[0091] The porosity of the diatomaceous earth or activated carbon is ≥90%.

[0092] Limit the immobilization conditions to pH (7.0–7.5) and shaking time (1–2 h). Neutral pH maintains bacterial activity, and shaking promotes bacterial diffusion into the pores of the carrier. Too long a shaking time may lead to bacterial autolysis, while too short a shaking time will result in insufficient adsorption.

[0093] Limiting the loading concentration (0.2×10) 7 ~19.7×10 7 The loading concentration (CFU / mL) and carrier porosity (≥90%) ensure sufficient microbial biomass for degradation, while the upper limit of loading concentration avoids local hypoxia caused by high concentration, which affects metabolic efficiency. A carrier with a porosity of ≥90% can provide a large specific surface area, increase the cell load, and at the same time ensure the mass transfer efficiency between substrate and oxygen.

[0094] In some embodiments, the volume ratio of the immobilized bacterial agent formed by Bacillus subtilis fixed on a porous carrier to the pretreated water sample is 0.1:1 to 0.2:1.

[0095] The volume ratio of immobilized bacterial agent to water sample is limited to 0.1:1 to 0.2:1. When the volume ratio is <0.1:1, the amount of microorganisms is insufficient, resulting in incomplete degradation of organic matter. When the volume ratio is >0.2:1, the excessive bacterial agent may consume too much dissolved oxygen, interfering with the accurate measurement of BOD5.

[0096] S4. Using the fully automated BOD5 analyzer, determine the initial dissolved oxygen and the endpoint dissolved oxygen after 5 days of inoculation of the inoculated water sample;

[0097] S5. Based on the initial dissolved oxygen and the endpoint dissolved oxygen, the five-day biochemical oxygen demand in the wastewater is obtained.

[0098] In dissolved oxygen (DO) measurement and BOD5 calculation, a fully automated analyzer accurately captures DO changes, and the oxygen demand is calculated based on the formula BOD5 = DO0 - DO5. Preliminary steps (sensor modification, sample pretreatment, and microbial immobilization) lay the foundation for DO measurement, ensuring that the difference between DO0 and DO5 accurately reflects the amount of oxygen consumed by organic matter degradation, avoiding calculation errors caused by instrument mistakes or abnormal reactions.

[0099] The detection method described in this application systematically eliminates the sources of error in traditional BOD5 detection through a four-level synergistic system of "sensor anti-interference optimization → sample purification and enrichment → microbial reaction standardization → precise signal acquisition," achieving accurate measurement of organic pollution levels in complex wastewater. The following is a detailed analysis of the synergistic effects between each step:

[0100] (1) Synergy between S1 (sensor modification) and S4 (dissolved oxygen measurement)

[0101] Anti-pollution and signal fidelity:

[0102] The superhydrophobic surface of the S1 fluorosilicone nanocoating (thickness 50-100nm, contact angle ≥110°) prevents suspended particles and microorganisms in wastewater from adhering to the electrode surface, thus avoiding increased oxygen mass transfer resistance caused by biofilm formation.

[0103] In S4, the modified sensor can capture the changes in DO caused by microbial metabolism in S3 in real time and accurately. Even in wastewater with high turbidity and high organic matter concentration, it can still maintain a measurement accuracy of ±0.1 mg / L, reducing the signal lag or drift caused by pollution in traditional sensors.

[0104] (2) Synergy between S2 (pretreatment) and S3 (microbial inoculation)

[0105] Removal of inhibitors and provision of substrates:

[0106] S2 modified activated carbon (specific surface area > 2000 m²) 2 / g) Removal of Hg through physical adsorption 2+Inhibitors such as humic acid (removal rate ≥95%) and porous silica gel (mass ratio 1:3) further adsorb small molecule toxins (such as phenols).

[0107] In S3, immobilized Bacillus subtilis (load concentration 0.2 × 10⁻⁶) 7 ~19.7×10 7 In an environment without inhibitors, the metabolic activity of microorganisms (CFU / mL) is enhanced. The concentration of degradable organic matter (such as glucose and fatty acids) increases due to the enrichment effect of S2, which increases the metabolic rate of microorganisms by 30% to 50% and the consumption of DO is more significant.

[0108] (3) Synergy between S2 (preprocessing) and S4 (measurement)

[0109] Optimize the matching of sample characteristics and measurement conditions:

[0110] The flow rate (1–3 mL / min) and dilution gradient (based on COD value) of the adsorption column in S2 are controlled to ensure that the BOD5 value of the pretreated water sample falls within the linear detection range of the instrument (0.5–4000 mg / L).

[0111] In S4, the modified sensor reduces the response time of the water sample with low turbidity (suspended solids SS < 10 mg / L after pretreatment) to < 30 seconds, which is more than 5 times faster than that of the untreated water sample (SS > 100 mg / L), thus reducing measurement delay error.

[0112] (4) Synergy between S3 (immobilized microorganisms) and S4 (measurement)

[0113] Stable reaction process and precise signal acquisition:

[0114] In S3, Bacillus subtilis immobilized on diatomaceous earth carrier (porosity ≥ 90%) forms a uniform biofilm, avoiding local concentration differences of free bacteria caused by water flow disturbance, thus stabilizing the DO consumption rate (RSD ≤ 5%).

[0115] In S4, the constant temperature culture system (20℃±0.5℃) works in conjunction with magnetic stirring to ensure that the immobilized microorganisms are in full contact with the substrate, and the DO measurement value can truly reflect the intensity of the biochemical reaction, reducing measurement fluctuations caused by uneven distribution of microorganisms.

[0116] (5) Triple synergy of S1 (sensor), S2 (pretreatment) and S3 (microorganisms)

[0117] Construct a complete anti-interference system:

[0118] The S1 fluorosilicone coating resists trace colloidal impurities remaining after pretreatment, the S2 deep purification reduces the frequency of sensor cleaning (from once a day to once a week), and the S3 immobilized microorganisms prevent excessive growth of biofilm on the sensor surface.

[0119] In summary, this application systematically solves the pain points of traditional BOD5 detection, such as low accuracy, poor repeatability, and weak anti-interference ability, through synergistic innovation in sensor modification, sample pretreatment, immobilized microorganism technology, and precision measurement. Its core advantages are as follows:

[0120] (1) Breakthrough improvement in sensor performance: The superhydrophobic nanocoating (50-100nm thickness, contact angle ≥110°) effectively resists the adhesion of organic matter and microorganisms, extending the sensor cleaning cycle from once a day to once a week, significantly reducing maintenance costs. Improved oxygen mass transfer efficiency: The nanoporous structure shortens the dissolved oxygen response time to <30 seconds (traditional sensors >150 seconds), achieving a measurement accuracy of ±0.1mg / L, with signal drift <3% in high-turbidity water samples.

[0121] (2) Optimization of sample pretreatment depth: Modified activated carbon-silica gel adsorption column (mass ratio 3:1) achieves dual function:

[0122] Remove inhibitors: Hg 2+ With an adsorption capacity ≥200mg / g and a humic acid removal rate ≥95%, it completely eliminates the inhibition of microorganisms by heavy metals and macromolecular organic matter; it enriches target substances: the retention rate of small molecule organic matter (such as acetic acid) is >90%, enhancing the signal of subsequent biochemical reactions by 30% to 50%. The intelligent dilution gradient (based on COD values ​​of 100–4000mg / L, diluted at 1:5 to 1:10) ensures that the BOD5 value of the water sample is always within the linear detection range of the instrument, avoiding errors caused by blind dilution in traditional methods.

[0123] (3) Standardization of the microbial reaction system: Immobilized Bacillus subtilis (load concentration 0.2×10⁻⁶) 7 ~19.7×10 7 CFU / mL, with a porosity ≥90% (carrier) achieves: Improved metabolic stability: DO consumption rate fluctuation RSD ≤5% (traditional inoculation method RSD ≥15%); Enhanced shock resistance: can withstand wastewater with COD up to 4000 mg / L, without the need for multiple dilutions, simplifying the operation process.

[0124] (4) Synergistic effect throughout the process: Triple anti-interference system: sensor coating resists residual impurities in pretreatment; deep purification of adsorption column reduces sensor contamination; immobilized microorganisms prevent excessive growth of biofilm. Improved detection accuracy: systematic error is reduced from ±20% of traditional methods to ±5%; parallel sample RSD ≤3%, repeatability is improved by more than 5 times; detection limit is reduced to 0.5 mg / L, suitable for low concentration water samples such as surface water.

[0125] (5) Application scenario expansion: adaptability to complex water samples: it can directly detect high-difficulty samples such as dyeing and printing wastewater, pharmaceutical wastewater, and chemical mixed wastewater without additional pretreatment; improved detection efficiency: combined with a fully automatic analyzer, the detection cycle of a single sample is shortened from 7 days in the traditional method to 5 days, and the throughput is increased by more than 30%.

[0126] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0127] Example 1

[0128] This embodiment provides a method for detecting biochemical oxygen demand (BOD) in wastewater. The wastewater is secondary biological effluent from a dyeing and printing factory, with initial indicators: COD: 350 mg / L, BOD5 (traditional dilution inoculation method): 92 mg / L, SS: 75 mg / L, Hg: 0.5 mg / L. 2+ The method described for 0.8 mg / L includes:

[0129] S11. A fluorinated organosilicon nano-coating is formed on the electrode surface of the dissolved oxygen sensor of the fully automatic BOD5 analyzer (model: OL4054) to obtain a fluorinated silicon modified dissolved oxygen sensor.

[0130] The fluorinated organosilicon nanocoating has a thickness of 70 nm and a contact angle of 115.4°.

[0131] The preparation method of the fluorinated organosilicon nanocoating includes: adding γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoethyl acrylate and acetic acid to an ethanol / water mixed solvent to carry out a hydrolysis-condensation reaction to obtain a first sol; adding nano-SiO2 particles to the first sol and dispersing them ultrasonically to obtain a second sol; adding methyl methacrylate and ammonium persulfate to the second sol to carry out a copolymerization reaction to obtain a coating; coating the coating onto the electrode surface of a dissolved oxygen sensor and thermally curing it to form a fluorinated organosilicon nanocoating.

[0132] The mass ratio of γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate, acetic acid, and ethanol / water (volume ratio 4:1) mixed solvent is 5:3:0.5:50. The parameters of the hydrolysis-condensation reaction include: reaction temperature of 70℃, reaction time of 3h, pH value adjusted to 5.0 by acetic acid; nano-SiO2 particle size of 10-50nm, added at 10% of the total mass of the first sol; ultrasonic dispersion power of 300W, time of 40min; molar ratio of methyl methacrylate to N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate of 3:1; ammonium persulfate added at 1.0% of the total mass of the second sol; copolymerization reaction temperature of 75℃, reaction time of 2.5h; thermosetting temperature of 130℃, time of 1.5h.

[0133] S21. The wastewater to be tested is pretreated in an adsorption column composed of modified activated carbon and porous silica gel to remove inhibitory substances and enrich degradable organic matter, so as to obtain a pretreated water sample.

[0134] The modified activated carbon to porous silica gel has a mass ratio of 3:1, and the dynamic adsorption flow rate of the adsorption column is 2 mL / min.

[0135] The modified activated carbon was prepared by KOH activation and has a specific surface area of ​​2207 m². 2 / g, for Hg 2+ The adsorption capacity is 202 mg / g, and the removal rate of humic acid is 95.5%.

[0136] S31. Bacillus subtilis (an existing strain, Bacillus subtilis CGMCC1.1086) was immobilized on a porous carrier and then added to the pretreated water sample to obtain an inoculated water sample.

[0137] The immobilization of Bacillus subtilis on a porous carrier includes: impregnating diatomaceous earth in Bacillus subtilis bacterial solution, adjusting the pH of the system to 7.2 with phosphate buffer, and shaking at a constant temperature for 1.5 hours to ensure that the bacteria are fully adsorbed into the pores of the carrier.

[0138] The loading concentration of Bacillus subtilis was 1×10⁻⁶. 7 CFU / mL, bacterial solution and diatomaceous earth are mixed at a volume ratio of 1:1;

[0139] The porosity of the diatomaceous earth is 95%.

[0140] The volume ratio of the immobilized bacterial agent formed by Bacillus subtilis fixed on a porous carrier to the pretreated water sample was 0.15:1.

[0141] The pretreated water sample is adjusted through the following steps:

[0142] pH adjustment: Adjust the pH of the water sample to 7 using phosphate buffer;

[0143] Dilute in a 1:8 gradient (COD = 43.75 mg / L).

[0144] S41. Using the fully automated BOD5 analyzer, determine the initial dissolved oxygen and the endpoint dissolved oxygen after 5 days of inoculation of the inoculated water sample;

[0145] S51. Based on the initial dissolved oxygen and the final dissolved oxygen, the five-day biochemical oxygen demand in the wastewater is obtained.

[0146] Test results: DO0 = 8.1 mg / L, DO5 = 3.2 mg / L, BOD5 = (8.1 - 3.2) × 8 = 39.2 mg / L, RSD (n = 5) = 2.4%.

[0147] Example 2

[0148] This embodiment provides a method for detecting biochemical oxygen demand (BOD) in wastewater. The wastewater sample is from an upstream section of a river, with the following initial parameters: COD: 85 mg / L, BOD5 (traditional dilution inoculation method): 4.5 mg / L, SS: 10 mg / L. The method includes:

[0149] S11. A fluorinated organosilicon nano-coating is formed on the electrode surface of the dissolved oxygen sensor of the fully automated BOD5 analyzer to obtain a fluorinated silicon modified dissolved oxygen sensor.

[0150] The fluorinated organosilicon nanocoating has a thickness of 50 nm and a contact angle of 110.2°.

[0151] The preparation method of the fluorinated organosilicon nanocoating includes: adding γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoethyl acrylate and acetic acid to an ethanol / water mixed solvent to carry out a hydrolysis-condensation reaction to obtain a first sol; adding nano-SiO2 particles to the first sol and dispersing them ultrasonically to obtain a second sol; adding methyl methacrylate and ammonium persulfate to the second sol to carry out a copolymerization reaction to obtain a coating; coating the coating onto the electrode surface of a dissolved oxygen sensor and thermally curing it to form a fluorinated organosilicon nanocoating.

[0152] The mass ratio of γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate, acetic acid, and ethanol / water (volume ratio 4:1) mixed solvent is 5:3:0.5:50. The parameters of the hydrolysis-condensation reaction include: reaction temperature of 60℃, reaction time of 4h, pH value adjusted to 4.0 by acetic acid; nano-SiO2 particle size of 10-50nm, added at 5% of the total mass of the first sol; ultrasonic dispersion power of 200W, time of 60min; molar ratio of methyl methacrylate to N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate of 2:1; ammonium persulfate added at 0.5% of the total mass of the second sol; copolymerization reaction temperature of 70℃, reaction time of 3h; thermosetting temperature of 120℃, time of 2h.

[0153] S21. The wastewater to be tested is pretreated in an adsorption column composed of modified activated carbon and porous silica gel to remove inhibitory substances and enrich degradable organic matter, so as to obtain a pretreated water sample.

[0154] The modified activated carbon to porous silica gel has a mass ratio of 3:1, and the dynamic adsorption flow rate of the adsorption column is 1 mL / min.

[0155] The modified activated carbon was prepared by KOH activation and has a specific surface area of ​​2000.5 m². 2 / g, for Hg 2+ The adsorption capacity was 203.7 mg / g, and the removal rate of humic acid was 95.4%.

[0156] S31. Bacillus subtilis (an existing strain, Bacillus subtilis CGMCC1.1086) was immobilized on a porous carrier and then added to the pretreated water sample to obtain an inoculated water sample.

[0157] The immobilization of Bacillus subtilis on a porous carrier includes: immersing a diatomaceous earth or activated carbon carrier in a Bacillus subtilis bacterial solution, adjusting the pH of the system to 7.0 using a phosphate buffer solution, and shaking at a constant temperature for 1 hour to ensure that the bacteria are fully adsorbed into the pores of the carrier.

[0158] The loading concentration of Bacillus subtilis was 0.2 × 10⁻⁶. 7 CFU / mL, bacterial solution and diatomaceous earth are mixed at a volume ratio of 1:1;

[0159] The porosity of the diatomaceous earth is 90%.

[0160] The volume ratio of the immobilized bacterial agent formed by Bacillus subtilis fixed on a porous carrier to the pretreated water sample was 0.1:1.

[0161] The pretreated water sample is adjusted through the following steps:

[0162] pH adjustment: Adjust the pH of the water sample to 6.5 using phosphate buffer;

[0163] The pretreated water sample is not diluted.

[0164] S41. Using the fully automated BOD5 analyzer, determine the initial dissolved oxygen and the endpoint dissolved oxygen after 5 days of inoculation of the inoculated water sample;

[0165] S51. Based on the initial dissolved oxygen and the final dissolved oxygen, the five-day biochemical oxygen demand in the wastewater is obtained.

[0166] Test results: DO0 = 8.3 mg / L, DO5 = 6.9 mg / L, BOD5 = 8.3 - 6.9 = 1.4 mg / L, RSD (n = 5) = 1.8%.

[0167] Example 3

[0168] This embodiment provides a method for detecting biochemical oxygen demand (BOD) in wastewater. The wastewater sample is a combined wastewater from a chemical plant. Initial indicators: COD: 3800 mg / L, BOD5 (traditional method): cannot be accurately measured (requires multiple dilutions), Hg 2+ 1.2 mg / L, the method includes:

[0169] S11. A fluorinated organosilicon nano-coating is formed on the electrode surface of the dissolved oxygen sensor of the fully automated BOD5 analyzer to obtain a fluorinated silicon modified dissolved oxygen sensor.

[0170] The fluorinated organosilicon nanocoating has a thickness of 100 nm and a contact angle of 120°.

[0171] The preparation method of the fluorinated organosilicon nanocoating includes: adding γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoethyl acrylate and acetic acid to an ethanol / water mixed solvent to carry out a hydrolysis-condensation reaction to obtain a first sol; adding nano-SiO2 particles to the first sol and dispersing them ultrasonically to obtain a second sol; adding methyl methacrylate and ammonium persulfate to the second sol to carry out a copolymerization reaction to obtain a coating; coating the coating onto the electrode surface of a dissolved oxygen sensor and thermally curing it to form a fluorinated organosilicon nanocoating.

[0172] The mass ratio of γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate, acetic acid, and ethanol / water (volume ratio 4:1) mixed solvent is 5:3:0.5:50. The parameters of the hydrolysis-condensation reaction include: reaction temperature of 80℃, reaction time of 2h, pH value adjusted to 6.0 by acetic acid; nano-SiO2 particle size of 10-50nm, added at 15% of the total mass of the first sol; ultrasonic dispersion power of 400W, time of 30min; molar ratio of methyl methacrylate to N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate of 4:1; ammonium persulfate added at 2.0% of the total mass of the second sol; copolymerization reaction temperature of 80℃, reaction time of 2h; thermosetting temperature of 150℃, time of 1h.

[0173] S21. The wastewater to be tested is pretreated in an adsorption column composed of modified activated carbon and porous silica gel to remove inhibitory substances and enrich degradable organic matter, so as to obtain a pretreated water sample.

[0174] The modified activated carbon and porous silica gel have a mass ratio of 3:1, and the dynamic adsorption flow rate of the adsorption column is 3 mL / min.

[0175] The modified activated carbon was prepared by KOH activation and has a specific surface area of ​​2000.1 m². 2 / g, for Hg 2+ The adsorption capacity was 200.6 mg / g, and the removal rate of humic acid was 95.3%.

[0176] S31. Bacillus subtilis (an existing strain, Bacillus subtilis CGMCC1.1086) was immobilized on a porous carrier and then added to the pretreated water sample to obtain an inoculated water sample.

[0177] The immobilization of Bacillus subtilis on a porous carrier includes: impregnating diatomaceous earth in Bacillus subtilis bacterial solution, adjusting the pH of the system to 7.5 with phosphate buffer, and shaking at a constant temperature for 1 hour to ensure that the bacteria are fully adsorbed into the pores of the carrier.

[0178] The loading concentration of Bacillus subtilis was 19.7 × 10⁻⁶. 7 CFU / mL, bacterial solution and diatomaceous earth are mixed at a volume ratio of 1:1;

[0179] The porosity of the diatomaceous earth is 90.3%.

[0180] The volume ratio of the immobilized bacterial agent formed by Bacillus subtilis fixed on a porous carrier to the pretreated water sample was 0.1:1 to 0.2:1.

[0181] The pretreated water sample is adjusted through the following steps:

[0182] pH adjustment: Adjust the pH of the water sample to 7.5 using phosphate buffer;

[0183] The water sample was serially diluted at a ratio of 1:10 (COD = 380 mg / L).

[0184] S41. Using the fully automated BOD5 analyzer, determine the initial dissolved oxygen and the endpoint dissolved oxygen after 5 days of inoculation of the inoculated water sample;

[0185] S51. Based on the initial dissolved oxygen and the final dissolved oxygen, the five-day biochemical oxygen demand in the wastewater is obtained.

[0186] Test results: DO0 = 8.0 mg / L, DO5 = 1.2 mg / L, BOD5 = (8.0 - 1.2) × 10 = 68 mg / L, RSD (n = 5) = 3.1%.

[0187] Comparative Example 1

[0188] This comparative example is based on the disclosure in Example 1, with the following modifications:

[0189] No fluorinated organosilicon nano-coating is formed on the electrode surface of the dissolved oxygen sensor; that is, a dissolved oxygen sensor without fluorinated organosilicon nano-coating is used directly.

[0190] Test results: The BOD5 measurement value fluctuated greatly, DO0 = 8.1 mg / L, DO5 = 4.5 mg / L, and the calculated BOD5 = (8.1-4.5)×8 = 28.8 mg / L, RSD (n=5) = 8.7%.

[0191] Comparative Example 1 failed to form a fluorinated organosilicon nanocoating on the surface of the dissolved oxygen sensor, resulting in the electrode being easily coated with suspended solids (SS = 75 mg / L) and Hg in wastewater. 2+ Organic matter adsorption and contamination can affect the accuracy of dissolved oxygen measurements. The hydrophobicity (contact angle 115.4°) and nanostructure of the fluorinated silicone coating can reduce contaminant adhesion, while the absence of this coating will reduce sensor response sensitivity and worsen data repeatability.

[0192] Comparative Example 2

[0193] This comparative example is based on the disclosure in Example 1, with the following modifications:

[0194] The wastewater to be tested is not pretreated in an adsorption column composed of modified activated carbon and porous silica gel.

[0195] Test results: BOD5 was significantly low, DO0 = 8.1 mg / L, DO5 = 6.8 mg / L, BOD5 = (8.1-6.8)×8 = 10.4 mg / L, RSD (n=5) = 5.3%.

[0196] Comparative Example 2 did not use modified activated carbon and porous silica gel for wastewater pretreatment, resulting in a higher Hg content in the wastewater. 2+ (0.8 mg / L) was not removed. Hg 2+ It is toxic to Bacillus subtilis, inhibiting the respiration of microorganisms and preventing them from effectively decomposing organic matter, thus reducing the consumption of dissolved oxygen. In addition, inhibitory substances such as humic acid in the wastewater were not removed, further reducing microbial activity and resulting in a significantly low BOD5 value.

[0197] Comparative Example 3

[0198] This comparative example is based on the disclosure in Example 1, with the following modifications:

[0199] Bacillus subtilis is not immobilized on a porous carrier; that is, Bacillus subtilis is directly inoculated into the pretreated water sample.

[0200] Test results: The BOD5 measurement value was low and the repeatability was poor. DO0 = 8.1 mg / L, DO5 = 5.6 mg / L, BOD5 = (8.1-5.6)×8 = 20.0 mg / L, RSD (n=5) = 10.2%.

[0201] In Comparative Example 3, Bacillus subtilis was not immobilized on a porous carrier. Direct inoculation resulted in bacterial aggregation in the water sample or loss with the water flow, leading to uneven microbial distribution. Immobilized carriers (such as diatomaceous earth with a porosity of 95%) provide a stable microenvironment, maintaining bacterial activity and promoting contact with organic matter. Free bacterial cells, on the other hand, exhibit faster activity decline during cultivation, reduced efficiency in degrading organic matter, decreased dissolved oxygen consumption, and increased data dispersion and RSD.

[0202] Comparative Example 4

[0203] This comparative example is based on the disclosure in Example 1, with the following modifications:

[0204] The wastewater to be tested is pretreated only in an adsorption column composed of modified activated carbon.

[0205] Test results: The BOD5 measurement value is low, DO0 = 8.1 mg / L, DO5 = 5.9 mg / L, and the calculated BOD5 = (8.1 - 5.9) × 8 = 17.6 mg / L, RSD (n = 5) ≈ 6.5%.

[0206] Reason: Although pretreatment with modified activated carbon alone can effectively remove Hg... 2+(Adsorption capacity 202 mg / g) and humic acid (removal rate 95.5%), but lacking the synergistic effect of porous silica gel. The high porosity (typically >80%) of porous silica gel can enrich small-molecule degradable organic matter, while activated carbon alone is selective in its adsorption of organic matter, favoring large molecules or non-polar substances, resulting in insufficient concentration of organic matter usable by Bacillus subtilis in the pretreated water sample. Furthermore, the polar surface of silica gel has a stronger adsorption capacity for polar organic matter; its absence leads to insufficient enrichment of organic matter, reduced dissolved oxygen consumed by microorganisms in decomposing organic matter, and consequently, a lower BOD5 measurement.

[0207] Comparative Example 5

[0208] This comparative example is based on the disclosure in Example 1, with the following modifications:

[0209] The wastewater to be tested is pretreated only in an adsorption column composed of porous silica gel.

[0210] Test results: The BOD5 measurement value was significantly low, such as DO0 = 8.1 mg / L, DO5 = 7.3 mg / L, BOD5 = (8.1-7.3)×8 = 6.4 mg / L, RSD (n=5)≈7.8%.

[0211] Reason: Using porous silica gel pretreatment alone cannot effectively remove Hg from wastewater. 2+ (0.8 mg / L). Hg 2+ Silica gel is highly toxic to Bacillus subtilis, inhibiting its respiratory enzyme activity and preventing the microorganism from decomposing organic matter. Simultaneously, silica gel's removal rate of inhibitory substances such as humic acid is far lower than that of modified activated carbon (activated carbon achieved a removal rate of 95.5% in the examples, while silica gel typically achieves <50%), and the residual inhibitors further weaken microbial activity. Furthermore, although silica gel can adsorb some organic matter, it lacks the high specific surface area (2207 m²) of activated carbon. 2 The microbial structure, with its abundant pores, results in insufficient adsorption capacity and selectivity for organic matter, leading to poor enrichment of degradable organic matter. In summary, inhibited microbial activity and limited available organic matter ultimately lead to a significant decrease in BOD5 levels.

[0212] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0213] Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0214] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for detecting biochemical oxygen demand (BOD) in wastewater, characterized in that, The method includes: A fluorinated organosilicon nano-coating was formed on the electrode surface of the dissolved oxygen sensor in a fully automated BOD5 analyzer to obtain a fluorinated silicon modified dissolved oxygen sensor. The wastewater to be tested was pretreated in an adsorption column composed of modified activated carbon and porous silica gel to remove inhibitory substances and enrich degradable organic matter, thus obtaining a pretreated water sample. Bacillus subtilis was immobilized on a porous carrier and then added to the pretreated water sample to obtain an inoculated water sample. The initial dissolved oxygen and the endpoint dissolved oxygen after 5 days of incubation were determined using the fully automated BOD5 analyzer. The five-day biochemical oxygen demand in wastewater is obtained based on the initial dissolved oxygen and the endpoint dissolved oxygen. The thickness of the fluorinated organosilicon nanocoating is 50–100 nm, and the contact angle is ≥110°. The mass ratio of the modified activated carbon to the porous silica gel is 3:1, and the dynamic adsorption flow rate of the adsorption column is 1-3 mL / min. The process of immobilizing Bacillus subtilis on a porous carrier includes: Diatomaceous earth or activated carbon carriers were impregnated in Bacillus subtilis bacterial solution. The pH of the system was adjusted to 7.0-7.5 with phosphate buffer and kept at a constant temperature and shaken for 1-2 hours to ensure that the bacteria were fully adsorbed into the carrier pores. The loading concentration of Bacillus subtilis was 0.2 × 10⁻⁶. 7 ~19.7×10 7 CFU / mL; the porosity of the diatomaceous earth or the activated carbon is ≥90%; The volume ratio of the immobilized bacterial agent formed by Bacillus subtilis fixed on the porous carrier to the pretreated water sample is 0.1:1 to 0.2:

1.

2. The method for detecting biochemical oxygen demand in wastewater according to claim 1, characterized in that, The preparation method of the fluorinated organosilicon nanocoating includes: γ-methacryloxypropyltrimethoxysilane, N-methylperfluorooctylsulfonylaminoacrylate ethyl acrylate and acetic acid were added to an ethanol / water mixed solvent to carry out a hydrolysis-condensation reaction to obtain the first sol; Nano-sized SiO2 particles were added to the first sol and dispersed by ultrasonication to obtain a second sol. Methyl methacrylate and ammonium persulfate are added to the second sol to carry out a copolymerization reaction to obtain a coating. The coating is applied to the electrode surface of the dissolved oxygen sensor and then thermally cured to form a fluorinated organosilicon nanocoating.

3. The method for detecting biochemical oxygen demand in wastewater according to claim 2, characterized in that, The parameters of the hydrolysis-condensation reaction include: a reaction temperature of 60–80°C, a reaction time of 2–4 h, and a pH value adjusted to 4.0–6.0 by acetic acid. The nano-SiO2 particles have a diameter of 10–50 nm and are added at a rate of 5–15% of the total mass of the first sol. The power of ultrasonic dispersion is 200-400W, and the time is 30-60min; The molar ratio of methyl methacrylate to N-methylperfluorooctyl sulfonyl amino acrylate is 2:1 to 4:1; The amount of ammonium persulfate added is 0.5% to 2.0% of the total mass of the second sol; The copolymerization reaction temperature is 70–80℃, and the reaction time is 2–3 hours. The temperature for heat curing is 120–150℃, and the time is 1–2 hours.

4. The method for detecting biochemical oxygen demand in wastewater according to claim 1, characterized in that, The modified activated carbon was prepared by KOH activation and has a specific surface area >2000 m². 2 / g, for Hg 2+ The adsorption capacity is ≥200mg / g, and the removal rate of humic acid is ≥95%.

5. The method for detecting biochemical oxygen demand in wastewater according to claim 1, characterized in that, The pretreated water sample is adjusted through the following steps: pH adjustment: Adjust the pH of the water sample to 6.5–7.5 using phosphate buffer solution; Dilution gradient: Based on the initial COD value of the water sample, no dilution is required when COD ≤ 100 mg / L, and when COD is 100–4000 mg / L, dilution is performed in a gradient of 1:5 to 1:

10. Sodium thiosulfate addition: Add sodium thiosulfate to the water sample containing residual chlorine to a final concentration of 10 mg / L, and let stand for 10 min to neutralize interference.

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