Method for detecting colonies in high-salt industrial recycled water

Abstract

The application discloses a detection method of colonies in high-salt industrial recycled water, and relates to the technical field of industrial water treatment and microorganism detection. The method comprises the following steps: step S1, dynamic sampling; step S2, sample pretreatment; step S3, inoculation; step S4, segmented culture; and step S5, colony counting and intelligent early warning. The culture medium used in the segmented culture is a composite salt-tolerant culture medium, which comprises 15-20 g / L of a basic culture medium, 8-12 g / L of a compatible solute compound, 1-2 g / L of a cell membrane repair agent, 5-8 g / L of a targeted nutrition system, 0.5-1 g / L of an anti-interference component and 2-3 g / L of a pH buffer, and deionized water is used as a solvent. The detection method has high detection rates under different salt concentrations and small relative deviations.

Description

Technical Field

[0001] This invention relates to the field of industrial water treatment and microbial detection technology, and in particular to a method for detecting bacterial colonies in high-salt industrial reclaimed water. Background Technology

[0002] With the increasing demand for water recycling, industrial recycled water is being used more and more widely in cooling, cleaning, and boiler feed. However, recycled water often contains high concentrations of inorganic salts (such as Cl⁻ and SO₄²⁻). 2 The presence of substances such as Na⁻ and Na⁺ creates a high osmotic pressure environment, which disrupts the osmotic pressure balance inside and outside microbial cells. This leads to cell dehydration and shrinkage, changes in cell membrane permeability, and inhibition of enzyme activity. Consequently, traditional culture media exhibit problems such as a significant decrease in colony detection rate, atypical colony morphology, and prolonged lag phase, failing to accurately reflect the level of microbial contamination.

[0003] Currently, common methods for detecting bacterial colonies in industrial reclaimed water mainly rely on monthly sampling or irregular sampling, and often use ordinary nutrient agar media. In high-salt environments, this can easily cause cell dehydration and rupture, failing to accurately reflect the number of viable bacteria in the system. Furthermore, existing detection methods suffer from various drawbacks, including poor media compatibility, rigid sampling strategies, crude sample pretreatment, unreasonable culture conditions, passive early warning mechanisms, cumbersome operations, and significant human error, making it difficult to meet the needs of high-frequency (e.g., weekly) monitoring. Although some studies have attempted to improve the culturability of high-salt samples by adding osmotic protectants, the lack of systematic pretreatment optimization and automated adaptation design still hinders the achievement of efficient, stable, and reproducible on-site detection.

[0004] Therefore, developing a colony detection method that is suitable for high-salt environments, allows for precise sampling, accurate detection, and timely early warning is of great significance for ensuring the safe reuse of high-salt industrial recycled water, extending equipment life, and stabilizing product quality. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a method for detecting bacterial colonies in high-salt industrial reclaimed water. This method enables a precise, efficient, periodic, and intelligent detection system for the total number of bacterial colonies in high-salt reclaimed water, thereby improving the operational safety and intelligent management level of industrial water systems.

[0006] To achieve the above objectives, the technical solution adopted by this invention is: a method for detecting bacterial colonies in high-salt industrial reclaimed water, comprising the following steps: Step S1, Dynamic Sampling: Monitor the salt concentration at key nodes of the high-salt industrial recycled water system in real time, and set a preset salt concentration threshold of 5000 mg / L; when the salt concentration is ≥5000 mg / L, the sampling frequency is adjusted to twice a week, and when the salt concentration is <5000 mg / L, the sampling frequency is once a week; collect 3-5 parallel samples each time, and the volume of each sample is 50-100 mL; Step S2, Sample Pretreatment: Filter each sample through a 0.45μm sterile filter membrane to remove particles larger than 0.45μm and insoluble organic matter; then sonicate the filtered sample in an ultrasonic bath for 3-5 minutes; take 25mL of the sonicated sample and add it to 225mL of sterile osmotic equilibrium solution to prepare a 1:10 initial dilution, and then serially dilute to form a 1:10 solution. -2 -10 -6 Diluted sample homogenate; Step S3, Inoculation: Immerse the test strip in the compound salt tolerance medium, take 1 mL of sample homogenate and drop it vertically into the center of the test strip, slowly cover the upper film and avoid the generation of air bubbles, use a pressure plate to gently press so that the sample solution evenly covers the culture area, and let it stand for 1-2 minutes to allow the medium to solidify. Step S4, Segmented Culture: First, culture in a constant temperature shaking incubator at 28±1℃ for 16±2h, then raise the temperature to 36±1℃ and culture for 32±2h, maintaining the humidity of the incubator at 60%-70% throughout the process; Step S5, Colony Counting and Intelligent Early Warning: After cultivation, the colonies turn red. The colony count is read using a colony counter. The test results, real-time salt concentration data, and production load data are input into a machine learning early warning model for intelligent early warning.

[0007] Preferably, the key nodes in step S1 include the outlet of the recycled water storage tank, the inlet of the cooling system, the water outlet for the cleaning process, and the peak value node for salt concentration monitoring.

[0008] Preferably, the ultrasonic treatment in step S2 has a power of 100-150W and a frequency of 20-30kHz.

[0009] Preferably, the osmotic balancing solution in step S2 comprises: 5 g / L tryptone, 3 g / L yeast extract, sodium chloride, 1.5 g / L glycine, 1.0 g / L betaine, 0.2 g / L MgSO4·7H2O, 0.1–0.3 g / L hydroxypropyl-β-cyclodextrin, 1–3 mg / L humic acid, and 2–5 mg / L reduced glutathione.

[0010] Preferably, the concentration of sodium chloride in step S2 is the salinity of the ultrasonically treated sample × (90%-110%).

[0011] Preferably, the composition of the composite salt-tolerant culture medium in step S3 is as follows: 15-20 g / L of basal culture medium, 8-12 g / L of compatible solute complex, 1-2 g / L of cell membrane repair agent, 5-8 g / L of targeted nutrient system, 0.5-1 g / L of anti-interference component, 2-3 g / L of pH buffer, deionized water as solvent, and pH of culture medium of 7.2±0.2.

[0012] Preferably, the basal culture medium is either LB medium or R2A agar medium.

[0013] Preferably, the compatible solute complex is a mixture of tetrahydropyrimidine, betaine, and trehalose in a mass ratio of 2:3:1.

[0014] Preferably, the cell membrane repair agent is a mixture of phosphatidylcholine and glutathione in a mass ratio of 1:1.

[0015] Preferably, the targeted nutrient system is a mixture of enzymatically hydrolyzed casein, yeast extract, MgSO4·7H2O, and MnCl2·4H2O in a mass ratio of 5:3:0.5:0.1; the enzymatically hydrolyzed casein is NZ-Case®Plus.

[0016] Preferably, the anti-interference component is a mixture of EDTA-2Na and bovine serum albumin at a mass ratio of 1:2; the pH buffer is HEPES buffer or PIPES buffer.

[0017] Preferably, the oscillation frequency of the constant temperature shaking incubator in step S4 is 70-90 rpm.

[0018] Preferably, the machine learning early warning model described in step S5 uses historical detection data, salt concentration data, and production condition data as training sets and is constructed using the random forest algorithm; the model outputs the risk level of microbial exceedance (low, medium, high). When the risk level is ≥ medium, the linkage disinfection system starts enhanced disinfection, automatically generates weekly and monthly reports, and supports mobile terminal push.

[0019] Due to the application of the above technical solution, the present invention has the following beneficial effects: (1) The method for detecting bacterial colonies in high-salt industrial reclaimed water disclosed in this invention achieves a balance between detection accuracy and efficiency through a dynamic sampling strategy. Traditional sampling often uses a fixed frequency, which is difficult to adapt to the large fluctuations in salt concentration of high-salt reclaimed water. However, this method dynamically adjusts the sampling frequency with a salt concentration threshold of 5000 mg / L. Key nodes cover core locations such as the outlet of the reclaimed water storage tank and the inlet of the cooling system. The design of collecting 3-5 parallel samples further reduces random errors and provides a more representative sample basis for subsequent detection.

[0020] (2) The method for detecting microbial colonies in high-salt industrial recycled water disclosed in this invention effectively improves the culturability of microorganisms under high-salt conditions through multi-dimensional optimization of the sample pretreatment process. After removing particulate interference by filtration through a 0.45μm filter membrane, ultrasonic treatment with a power of 100-150W can disperse the aggregated microbial colonies and avoid counting deviations. The precise matching of sodium chloride concentration in the osmotic equilibrium solution with the sample salinity (90%-110%), combined with the synergistic effects of osmotic protectants such as glycine and betaine, as well as reduced glutathione, hydroxypropyl-β-cyclodextrin, and humic acid, effectively alleviates the damage to microbial cell membranes caused by high osmotic pressure and solves the core problem of microbial dehydration and inactivation in traditional pretreatment.

[0021] (3) The method for detecting colonies in high-salt industrial recycled water disclosed in this invention significantly improves the colony detection rate and morphological typicality through the customized formulation of the composite salt-tolerant culture medium. Compared with ordinary nutrient agar, the tetrahydropyrimidine-betaine-trehalose composite compatible solute in this culture medium can stabilize cell osmotic pressure, the phosphatidylcholine-glutathione repair agent can repair damaged cell membranes, the targeted nutrient system mainly composed of enzymatic hydrolysis of casein can adapt to the metabolic needs of high-salt-adaptive microorganisms, and the anti-interference combination of EDTA-2Na and bovine serum albumin reduces the interference of high-salt matrix on detection, making the colony morphology clearer and the counting more accurate.

[0022] (4) The method for detecting bacterial colonies in high-salt industrial recycled water disclosed in this invention adopts a segmented culture process that takes into account both the needs of microbial recovery and efficient proliferation. Low-temperature shaking culture at 28±1℃ (70-90rpm) for 16±2h can promote the recovery and repair of damaged microorganisms, while high-temperature culture at 36±1℃ for 32±2h accelerates microbial proliferation. The constant humidity environment of 60%-70% ensures the integrity of colony morphology. This gradient temperature design solves the problems of excessively long lag period and slow colony growth in traditional single-temperature culture, and improves colony detection efficiency while shortening the culture cycle. The intelligent early warning system realizes the closed-loop linkage between detection and risk control. The machine learning model built based on the random forest algorithm integrates multi-dimensional parameters such as historical detection data, salt concentration and production load. The low, medium and high risk levels output can be directly linked to the disinfection system. Medium and higher risks automatically start enhanced disinfection. The automatic generation of weekly and monthly reports and the mobile terminal push function break the passive early warning mode of traditional detection and improve the intelligent level of industrial water system management.

[0023] (5) The method for detecting bacterial colonies in high-salinity industrial reclaimed water disclosed in this invention is designed with both operability and stability in mind, and is adapted to the needs of high-frequency monitoring. From the automated adaptation of dynamic sampling to the standardized operation of pretreatment and inoculation, and the intelligent linkage of colony counting and early warning, the cumbersome process of traditional detection is greatly simplified and human error is reduced. Compared with the traditional monthly sampling mode, this method can stably support high-frequency monitoring 1-2 times per week, providing a continuous and reliable technical guarantee for the safe reuse of high-salinity industrial reclaimed water.

[0024] (6) The method for detecting bacterial colonies in high-salt industrial recycled water disclosed in this invention comprises the following components: tryptone 5 g / L, yeast extract 3 g / L, sodium chloride, glycine 1.5 g / L, betaine 1.0 g / L, MgSO4·7H2O 0.2 g / L, hydroxypropyl-β-cyclodextrin 0.1–0.3 g / L, humic acid 1–3 mg / L, and reduced glutathione 2–5 mg / L; the concentration of sodium chloride is the salinity of the sample after ultrasonic treatment × (90%–110%); the composition of the composite salt-tolerant culture medium is: basal culture medium 15–20 g / L, compatible solute complex 8–12 g / L, cell membrane repair agent 1–2 g / L, targeted nutrient system 5–8 g / L, anti-interference component 0.5–1 g / L, pH buffer 2–3 g / L, deionized water as solvent, and pH of the culture medium 7.2 ± 0.2. Through the synergistic effect of its components, this detection method achieves high detection rates and low relative deviations at different salt concentrations. It enables precise, efficient, periodic, and intelligent detection of total bacterial counts in high-salt reclaimed water, improving the operational safety and intelligent management of industrial water systems. In the high-salt industrial reclaimed water bacterial colony detection system, hydroxypropyl-β-cyclodextrin (HP-β-CD) and humic acid work synergistically across three dimensions: toxicity removal, nutrient supply, and environmental stability. This specifically addresses the three core problems faced by microorganisms in high-salt environments: inhibition by organic pollutants, nutrient deficiency, and fluctuations in the living environment. This provides crucial support for microbial survival and proliferation, ultimately improving the colony detection rate and detection stability. Detailed Implementation

[0025] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0026] Example 1 A method for detecting bacterial colonies in high-salt industrial recycled water, comprising the following steps: Step S1, Dynamic Sampling: Monitor the salt concentration at key nodes of the high-salt industrial recycled water system in real time, and set the preset salt concentration threshold to 5000 mg / L; when the salt concentration is ≥5000 mg / L, the sampling frequency is adjusted to twice a week, and when the salt concentration is <5000 mg / L, the sampling frequency is once a week; collect 3 parallel samples each time, and each sample volume is 50 mL. Step S2, Sample Pretreatment: Filter each sample through a 0.45μm sterile filter membrane to remove particles larger than 0.45μm and insoluble organic matter; then sonicate the filtered sample in an ultrasonic bath for 3 minutes; take 25mL of the sonicated sample and add it to 225mL of sterile osmotic equilibrium solution to prepare a 1:10 initial dilution, and then serially dilute to form a 1:10 solution. -2 -10 -6 Diluted sample homogenate; Step S3, Inoculation: Immerse the test strip in the compound salt tolerance medium, take 1 mL of sample homogenate and drop it vertically into the center of the test strip, slowly cover the upper film and avoid the generation of air bubbles, use a pressure plate to gently press so that the sample solution evenly covers the culture area, and let it stand for 1 min to solidify the medium. Step S4, Segmented Culture: First, culture at 28℃ for 16 hours in a constant temperature shaking incubator, then raise the temperature to 36℃ and culture for 32 hours, maintaining the humidity of the incubator at 60% throughout the process; Step S5, Colony Counting and Intelligent Early Warning: After cultivation, the colonies turn red. The colony count is read using a colony counter. The test results, real-time salt concentration data, and production load data are input into a machine learning early warning model for intelligent early warning.

[0027] The key nodes mentioned in step S1 include the outlet of the recycled water storage tank, the inlet of the cooling system, the water outlet for the cleaning process, and the peak value node for salt concentration monitoring; the ultrasonic treatment mentioned in step S2 has a power of 100W and a frequency of 20kHz.

[0028] The osmotic equilibrium solution in step S2 consists of: 5 g / L tryptone, 3 g / L yeast extract, sodium chloride, 1.5 g / L glycine, 1.0 g / L betaine, 0.2 g / L MgSO4·7H2O, 0.1 g / L hydroxypropyl-β-cyclodextrin, 1 mg / L humic acid, and 2 mg / L reduced glutathione; the concentration of sodium chloride is 90% of the salinity of the ultrasonically treated sample.

[0029] The composition of the composite salt-tolerant culture medium in step S3 is as follows: 15 g / L basal medium, 8 g / L compatible solute complex, 1 g / L cell membrane repair agent, 5 g / L targeted nutrient system, 0.5 g / L anti-interference component, and 2 g / L pH buffer. The solvent is deionized water, and the pH of the culture medium is 7.2. The basal medium is LB medium. The compatible solute complex is a mixture of tetrahydropyrimidine, betaine, and trehalose in a mass ratio of 2:3:1. The cell membrane... The repair agent is a compound of phosphatidylcholine and glutathione in a mass ratio of 1:1; the targeted nutrient system is a compound of enzymatically hydrolyzed casein, yeast extract, MgSO4·7H2O, and MnCl2·4H2O in a mass ratio of 5:3:0.5:0.1; the enzymatically hydrolyzed casein is NZ-Case®Plus; the anti-interference component is a compound of EDTA-2Na and bovine serum albumin in a mass ratio of 1:2; and the pH buffer is HEPES buffer.

[0030] The oscillation frequency of the constant temperature shaking incubator mentioned in step S4 is 70 rpm; the machine learning early warning model mentioned in step S5 uses historical detection data, salt concentration data, and production condition data as training sets and is constructed using the random forest algorithm; the model outputs the risk level of microbial exceedance (low, medium, high). When the risk level is ≥ medium, the linkage disinfection system starts enhanced disinfection, automatically generates weekly and monthly reports, and supports mobile terminal push.

[0031] Example 2 A method for detecting bacterial colonies in high-salt industrial recycled water, comprising the following steps: Step S1, Dynamic Sampling: Monitor the salt concentration at key nodes of the high-salt industrial recycled water system in real time, and set the preset salt concentration threshold to 5000 mg / L; when the salt concentration is ≥5000 mg / L, the sampling frequency is adjusted to twice a week, and when the salt concentration is <5000 mg / L, the sampling frequency is once a week; collect 4 parallel samples each time, and each sample volume is 60 mL. Step S2, Sample Pretreatment: Filter each sample through a 0.45μm sterile filter membrane to remove particles >0.45μm and insoluble organic matter; then sonicate the filtered sample in an ultrasonic bath for 3.5 min; take 25mL of the sonicated sample and add it to 225mL of sterile osmotic equilibrium solution to prepare a 1:10 initial dilution, and then serially dilute to form a 1:10 solution. -2 -10 -6 Diluted sample homogenate; Step S3, Inoculation: Immerse the test strip in the compound salt tolerance medium, take 1 mL of sample homogenate and drop it vertically into the center of the test strip, slowly cover the upper film and avoid the generation of air bubbles, use a pressure plate to gently press so that the sample solution evenly covers the culture area, and let it stand for 1.2 min to solidify the medium. Step S4, Segmented Culture: First, culture at 28℃ for 16 hours in a constant temperature shaking incubator, then raise the temperature to 36℃ and culture for 32 hours, maintaining the humidity of the incubator at 63% throughout the process; Step S5, Colony Counting and Intelligent Early Warning: After cultivation, the colonies turn red. The colony count is read using a colony counter. The test results, real-time salt concentration data, and production load data are input into a machine learning early warning model for intelligent early warning.

[0032] The key nodes mentioned in step S1 include the outlet of the recycled water storage tank, the inlet of the cooling system, the water outlet for the cleaning process, and the peak value node for salt concentration monitoring; the ultrasonic treatment in step S2 has a power of 120W and a frequency of 23kHz; the composition of the osmotic equilibrium solution in step S2 is: 5g / L tryptone, 3g / L yeast extract, sodium chloride, 1.5g / L glycine, 1.0g / L betaine, 0.2g / L MgSO4·7H2O, 0.15g / L hydroxypropyl-β-cyclodextrin, 1.5mg / L humic acid, and 3mg / L reduced glutathione; the concentration of sodium chloride is 95% of the salinity of the ultrasonically treated sample.

[0033] The composition of the composite salt-tolerant culture medium in step S3 is as follows: 16 g / L basal medium, 9 g / L compatible solute complex, 1.2 g / L cell membrane repair agent, 6 g / L targeted nutrient system, 0.7 g / L anti-interference component, and 2.3 g / L pH buffer, with deionized water as the solvent and a pH of 7; the basal medium is R2A agar medium; the compatible solute complex is a mixture of tetrahydropyrimidine, betaine, and trehalose in a mass ratio of 2:3:1. The cell membrane repair agent is a mixture of phosphatidylcholine and glutathione in a 1:1 mass ratio; the targeted nutrient system is a mixture of enzymatically hydrolyzed casein, yeast extract, MgSO4·7H2O, and MnCl2·4H2O in a 5:3:0.5:0.1 mass ratio; the enzymatically hydrolyzed casein is NZ-Case®Plus; the anti-interference component is a mixture of EDTA-2Na and bovine serum albumin in a 1:2 mass ratio; and the pH buffer is PIPES buffer.

[0034] The oscillation frequency of the constant temperature shaking incubator mentioned in step S4 is 75 rpm; the machine learning early warning model mentioned in step S5 uses historical detection data, salt concentration data, and production condition data as training sets and is constructed using the random forest algorithm; the model outputs the risk level of microbial exceedance (low, medium, high). When the risk level is ≥ medium, the linkage disinfection system starts enhanced disinfection, automatically generates weekly and monthly reports, and supports mobile terminal push.

[0035] Example 3 A method for detecting bacterial colonies in high-salt industrial recycled water, comprising the following steps: Step S1, Dynamic Sampling: Monitor the salt concentration at key nodes of the high-salt industrial recycled water system in real time, and set the preset salt concentration threshold to 5000 mg / L; when the salt concentration is ≥5000 mg / L, the sampling frequency is adjusted to twice a week, and when the salt concentration is <5000 mg / L, the sampling frequency is once a week; collect 3-5 parallel samples each time, and each sample volume is 80 mL; Step S2, Sample Pretreatment: Filter each sample through a 0.45μm sterile filter membrane to remove particles larger than 0.45μm and insoluble organic matter; then sonicate the filtered sample in an ultrasonic bath for 4 minutes; take 25mL of the sonicated sample and add it to 225mL of sterile osmotic equilibrium solution to prepare a 1:10 initial dilution, and then serially dilute to form a 1:10 solution. -2 -10 -6 Diluted sample homogenate; Step S3, Inoculation: Immerse the test strip in the compound salt tolerance medium, take 1 mL of sample homogenate and drop it vertically into the center of the test strip, slowly cover the upper film and avoid the generation of air bubbles, use a pressure plate to gently press so that the sample solution evenly covers the culture area, and let it stand for 1.5 min to solidify the medium. Step S4, Segmented Culture: First, culture at 29℃ for 17 hours in a constant temperature shaking incubator, then raise the temperature to 37℃ and culture for 33 hours, maintaining the humidity of the incubator at 65% throughout the process; Step S5, Colony Counting and Intelligent Early Warning: After cultivation, the colonies turn red. The colony count is read using a colony counter. The test results, real-time salt concentration data, and production load data are input into a machine learning early warning model for intelligent early warning.

[0036] The key nodes mentioned in step S1 include the outlet of the recycled water storage tank, the inlet of the cooling system, the water outlet for the cleaning process, and the peak value node for salt concentration monitoring; the ultrasonic treatment in step S2 has a power of 130W and a frequency of 25kHz; the composition of the osmotic equilibrium solution in step S2 is: 5g / L tryptone, 3g / L yeast extract, sodium chloride, 1.5g / L glycine, 1.0g / L betaine, 0.2g / L MgSO4·7H2O, 0.2g / L hydroxypropyl-β-cyclodextrin, 2mg / L humic acid, and 3.5mg / L reduced glutathione; the concentration of sodium chloride in step S2 is 100% of the salinity of the ultrasonically treated sample.

[0037] The composite salt-tolerant culture medium in step S3 consists of: 18 g / L basal medium, 10 g / L compatible solute complex, 1.5 g / L cell membrane repair agent, 6.5 g / L targeted nutrient system, 0.8 g / L anti-interference component, and 2.5 g / L pH buffer. The solvent is deionized water, and the pH of the culture medium is 7.3. The basal medium is LB medium. The compatible solute complex is a mixture of tetrahydropyrimidine, betaine, and trehalose in a mass ratio of 2:3:1. The cell membrane repair agent... The compound is composed of phosphatidylcholine and glutathione in a mass ratio of 1:1; the targeted nutrient system is composed of enzymatically hydrolyzed casein, yeast extract, MgSO4·7H2O, and MnCl2·4H2O in a mass ratio of 5:3:0.5:0.1; the enzymatically hydrolyzed casein is NZ-Case®Plus; the anti-interference component is composed of EDTA-2Na and bovine serum albumin in a mass ratio of 1:2; the pH buffer is HEPES buffer or PIPES buffer.

[0038] The oscillation frequency of the constant temperature shaking incubator mentioned in step S4 is 80 rpm; the machine learning early warning model mentioned in step S5 uses historical detection data, salt concentration data, and production condition data as training sets and is constructed using the random forest algorithm; the model outputs the risk level of microbial exceedance (low, medium, high). When the risk level is ≥ medium, the linkage disinfection system starts enhanced disinfection, automatically generates weekly and monthly reports, and supports mobile terminal push.

[0039] Example 4 A method for detecting bacterial colonies in high-salt industrial recycled water, comprising the following steps: Step S1, Dynamic Sampling: Monitor the salt concentration at key nodes of the high-salt industrial recycled water system in real time, and set the preset salt concentration threshold to 5000 mg / L; when the salt concentration is ≥5000 mg / L, the sampling frequency is adjusted to twice a week, and when the salt concentration is <5000 mg / L, the sampling frequency is once a week; collect 5 parallel samples each time, and each sample volume is 90 mL. Step S2, Sample Pretreatment: Filter each sample through a 0.45μm sterile filter membrane to remove particles larger than 0.45μm and insoluble organic matter; then sonicate the filtered sample in an ultrasonic bath for 4.5 min; take 25mL of the sonicated sample and add it to 225mL of sterile osmotic equilibrium solution to prepare a 1:10 initial dilution, and then serially dilute to form a 1:10 solution. -2 -10 -6 Diluted sample homogenate; Step S3, Inoculation: Immerse the test strip in the compound salt tolerance medium, take 1 mL of sample homogenate and drop it vertically into the center of the test strip, slowly cover the upper film and avoid the generation of air bubbles, use a pressure plate to gently press so that the sample solution evenly covers the culture area, and let it stand for 1.8 min to allow the culture medium to solidify. Step S4, Segmented Culture: First, culture at 28℃ for 18 hours in a constant temperature shaking incubator, then raise the temperature to 36℃ and culture for 34 hours, maintaining the humidity of the incubator at 68% throughout the process; Step S5, Colony Counting and Intelligent Early Warning: After cultivation, the colonies turn red. The colony count is read using a colony counter. The test results, real-time salt concentration data, and production load data are input into a machine learning early warning model for intelligent early warning.

[0040] The key nodes mentioned in step S1 include the outlet of the recycled water storage tank, the inlet of the cooling system, the water outlet for the cleaning process, and the peak value node for salt concentration monitoring; the ultrasonic treatment in step S2 has a power of 140W and a frequency of 28kHz; the composition of the osmotic equilibrium solution in step S2 is: 5g / L tryptone, 3g / L yeast extract, sodium chloride, 1.5g / L glycine, 1.0g / L betaine, 0.2g / L MgSO4·7H2O, 0.25g / L hydroxypropyl-β-cyclodextrin, 2.5mg / L humic acid, and 4.5mg / L reduced glutathione; the concentration of sodium chloride in step S2 is 105% of the salinity of the ultrasonically treated sample.

[0041] The composite salt tolerance culture medium in step S3 consists of: 19 g / L basal medium, 11 g / L compatible solute complex, 1.8 g / L cell membrane repair agent, 7.5 g / L targeted nutrient system, 0.9 g / L anti-interference component, and 2.8 g / L pH buffer. The solvent is deionized water, and the pH of the culture medium is 7.2. The basal medium is R2A agar medium. The compatible solute complex is a mixture of tetrahydropyrimidine, betaine, and trehalose in a mass ratio of 2:3:1. The cell membrane repair agent is a mixture of phosphatidylcholine and glutathione in a 1:1 mass ratio; the targeted nutrient system is a mixture of enzymatically hydrolyzed casein, yeast extract, MgSO4·7H2O, and MnCl2·4H2O in a 5:3:0.5:0.1 mass ratio; the enzymatically hydrolyzed casein is NZ-Case®Plus; the anti-interference component is a mixture of EDTA-2Na and bovine serum albumin in a 1:2 mass ratio; and the pH buffer is HEPES buffer.

[0042] The oscillation frequency of the constant temperature shaking incubator mentioned in step S4 is 85 rpm; the machine learning early warning model mentioned in step S5 uses historical detection data, salt concentration data, and production condition data as training sets and is constructed using the random forest algorithm; the model outputs the risk level of microbial exceedance (low, medium, high). When the risk level is ≥ medium, the linkage disinfection system starts enhanced disinfection, automatically generates weekly and monthly reports, and supports mobile terminal push.

[0043] Example 5 A method for detecting bacterial colonies in high-salt industrial recycled water, comprising the following steps: Step S1, Dynamic Sampling: Monitor the salt concentration at key nodes of the high-salt industrial recycled water system in real time, and set the preset salt concentration threshold to 5000 mg / L; when the salt concentration is ≥5000 mg / L, the sampling frequency is adjusted to twice a week, and when the salt concentration is <5000 mg / L, the sampling frequency is once a week; collect 3-5 parallel samples each time, and the volume of each sample is 100 mL; Step S2, Sample Pretreatment: Filter each sample through a 0.45μm sterile filter membrane to remove particles larger than 0.45μm and insoluble organic matter; then sonicate the filtered sample in an ultrasonic bath for 5 minutes; take 25mL of the sonicated sample and add it to 225mL of sterile osmotic equilibrium solution to prepare a 1:10 initial dilution, and then serially dilute to form a 1:10 solution. -2 -10 -6 Diluted sample homogenate; Step S3, Inoculation: Immerse the test strip in the compound salt tolerance medium, take 1 mL of sample homogenate and drop it vertically into the center of the test strip, slowly cover the upper film and avoid the generation of air bubbles, use a pressure plate to gently press so that the sample solution evenly covers the culture area, and let it stand for 2 minutes to allow the medium to solidify. Step S4, Segmented Culture: First, culture at 28℃ for 16 hours in a constant temperature shaking incubator, then raise the temperature to 36℃ and culture for 32 hours, maintaining the humidity of the incubator at 60% throughout the process; Step S5, Colony Counting and Intelligent Early Warning: After cultivation, the colonies turn red. The colony count is read using a colony counter. The test results, real-time salt concentration data, and production load data are input into a machine learning early warning model for intelligent early warning.

[0044] The key nodes mentioned in step S1 include the outlet of the recycled water storage tank, the inlet of the cooling system, the water outlet for the cleaning process, and the peak value node for salt concentration monitoring; the ultrasonic treatment in step S2 has a power of 150W and a frequency of 30kHz; the composition of the osmotic equilibrium solution in step S2 is: 5g / L tryptone, 3g / L yeast extract, sodium chloride, 1.5g / L glycine, 1.0g / L betaine, 0.2g / L MgSO4·7H2O, 0.3g / L hydroxypropyl-β-cyclodextrin, 3mg / L humic acid, and 5mg / L reduced glutathione; the concentration of sodium chloride in step S2 is 110% of the salinity of the ultrasonically treated sample.

[0045] The composite salt-tolerant culture medium in step S3 consists of: 20 g / L basal medium, 12 g / L compatible solute complex, 2 g / L cell membrane repair agent, 8 g / L targeted nutrient system, 1 g / L anti-interference component, and 3 g / L pH buffer. The solvent is deionized water, and the pH of the culture medium is 7.2. The basal medium is LB medium. The compatible solute complex is a mixture of tetrahydropyrimidine, betaine, and trehalose in a mass ratio of 2:3:1. The cell membrane repair agent is phosphorus... Acylcholine and glutathione are compounded in a mass ratio of 1:1; the targeted nutrient system is compounded in a mass ratio of 5:3:0.5:0.1 of enzymatically hydrolyzed casein, yeast extract, MgSO4·7H2O, and MnCl2·4H2O; the enzymatically hydrolyzed casein is NZ-Case®Plus; the anti-interference component is compounded in a mass ratio of 1:2 of EDTA-2Na and bovine serum albumin; the pH buffer is HEPES buffer or PIPES buffer.

[0046] The oscillation frequency of the constant temperature shaking incubator mentioned in step S4 is 90 rpm; the machine learning early warning model mentioned in step S5 uses historical detection data, salt concentration data, and production condition data as training sets and is constructed using the random forest algorithm; the model outputs the risk level of microbial exceedance (low, medium, high). When the risk level is ≥ medium, the linkage disinfection system starts enhanced disinfection, automatically generates weekly and monthly reports, and supports mobile terminal push.

[0047] Comparative Example 1 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that hydroxypropyl-β-cyclodextrin and humic acid are not added.

[0048] Comparative Example 2 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that an equal amount of physiological saline is used instead of the osmotic balance solution.

[0049] Comparative Example 3 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that no compatible solute complex is added.

[0050] Comparative Example 4 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that no anti-interference components are added.

[0051] Comparative Example 5 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that an equal amount of phosphatidylcholine is used instead of glutathione.

[0052] Comparative Example 6 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that an equal amount of glutathione is used instead of phosphatidylcholine.

[0053] Comparative Example 7 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that an equal amount of hydroxypropyl-β-cyclodextrin is used instead of humic acid.

[0054] Comparative Example 8 This example provides a method for detecting bacterial colonies in high-salt industrial recycled water, which is basically the same as that in Example 5, except that an equal amount of humic acid is used instead of hydroxypropyl-β-cyclodextrin.

[0055] To further illustrate the beneficial technical effects of the detection method for bacterial colonies in high-salinity industrial reclaimed water involved in the embodiments of this application, relevant performance tests were conducted on the detection methods for bacterial colonies in high-salinity industrial reclaimed water involved in Example 5 and Comparative Examples 1-8. The test results are shown in Table 1, and the test methods are as follows: High-salt industrial recycled water samples: taken from the recycled water system of a petrochemical enterprise, with salt concentrations of 3000 mg / L (sample A), 8000 mg / L (sample B), and 12000 mg / L (sample C). Each sample was tested according to the methods described in the examples. Sample A was serially diluted to 10⁻⁻⁶. 3 Sample B was serially diluted to 10⁻ 4 C sample serially diluted to 10⁻ 5 The detection rate and relative deviation were statistically analyzed and calculated.

[0056] Table 1. Results of bacterial colony test in high-salt industrial recycled water As shown in Table 1, the detection rate of bacterial colonies in the high-salt industrial recycled water prepared in Example 5 of the present invention is ≥98.1% at different salt concentrations, with a relative deviation of ≤4.2%, which is significantly better than the comparative example. The combined use of hydroxypropyl-β-cyclodextrin, humic acid, osmotic equilibrium solution, compatible solute complex, interfering components, phosphatidylcholine and glutathione is beneficial to improving the above performance.

[0057] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting bacterial colonies in high-salt industrial recycled water, characterized in that, Includes the following steps: Step S1, Dynamic Sampling: Monitor the salt concentration at key nodes of the high-salt industrial recycled water system in real time, and set a preset salt concentration threshold of 5000 mg / L; when the salt concentration is ≥5000 mg / L, the sampling frequency is adjusted to twice a week, and when the salt concentration is <5000 mg / L, the sampling frequency is once a week; collect 3-5 parallel samples each time, and the volume of each sample is 50-100 mL; Step S2, Sample Pretreatment: Filter each sample through a 0.45μm sterile filter membrane to remove particles larger than 0.45μm and insoluble organic matter; then sonicate the filtered sample in an ultrasonic bath for 3-5 minutes; take 25mL of the sonicated sample and add it to 225mL of sterile osmotic equilibrium solution to prepare a 1:10 initial dilution, and then serially dilute to form a 1:10 solution. -2 -10 -6 The diluted sample was homogenized; the osmotic equilibrium solution consisted of: 5 g / L tryptone, 3 g / L yeast extract, sodium chloride, 1.5 g / L glycine, 1.0 g / L betaine, 0.2 g / L MgSO4·7H2O, 0.1-0.3 g / L hydroxypropyl-β-cyclodextrin, 1-3 mg / L humic acid, and 2-5 mg / L reduced glutathione; the concentration of sodium chloride was (90%-110%) of the salinity of the ultrasonically treated sample. Step S3, Inoculation: Immerse the test strip in the composite salt tolerance medium. Add 1 mL of sample homogenate vertically to the center of the test strip, slowly covering the upper membrane while avoiding air bubbles. Gently press with a pressure plate to evenly cover the culture area with the sample solution. Let stand for 1-2 minutes to allow the medium to solidify. The composite salt tolerance medium consists of: 15-20 g / L basal medium, 8-12 g / L compatible solute complex, 1-2 g / L cell membrane repair agent, 5-8 g / L targeted nutrient system, 0.5-1 g / L anti-interference component, and 2-3 g / L pH buffer. The solvent is deionized water. The pH of the medium is... The value is 7.2 ± 0.2; the basal culture medium is any one of LB medium and R2A agar medium; the compatibility solute complex is a mixture of tetrahydropyrimidine, betaine, and trehalose in a mass ratio of 2:3:1; the cell membrane repair agent is a mixture of phosphatidylcholine and glutathione in a mass ratio of 1:1; the targeted nutrient system is a mixture of enzymatically hydrolyzed casein, yeast extract, MgSO4·7H2O, and MnCl2·4H2O in a mass ratio of 5:3:0.5:0.1; the anti-interference component is a mixture of EDTA-2Na and bovine serum albumin in a mass ratio of 1:

2. Step S4, Segmented Culture: First, culture in a constant temperature shaking incubator at 28±1℃ for 16±2h, then raise the temperature to 36±1℃ and culture for 32±2h, maintaining the humidity of the incubator at 60%-70% throughout the process; Step S5, Colony Counting and Intelligent Early Warning: After cultivation, the colonies turn red. The colony count is read using a colony counter. The test results, real-time salt concentration data, and production load data are input into a machine learning early warning model for intelligent early warning.

2. The method for detecting bacterial colonies in high-salt industrial reclaimed water according to claim 1, characterized in that, The key nodes mentioned in step S1 include the outlet of the recycled water storage tank, the inlet of the cooling system, the water outlet for the cleaning process, and the peak value node for salt concentration monitoring.

3. The method for detecting bacterial colonies in high-salt industrial reclaimed water according to claim 1, characterized in that, The ultrasonic treatment in step S2 has a power of 100-150W and a frequency of 20-30kHz.

4. The method for detecting bacterial colonies in high-salt industrial reclaimed water according to claim 1, characterized in that, The pH buffer is either HEPES buffer or PIPES buffer.

5. The method for detecting bacterial colonies in high-salt industrial reclaimed water according to claim 1, characterized in that, The oscillation frequency of the constant temperature shaking incubator mentioned in step S4 is 70-90 rpm.

6. The method for detecting bacterial colonies in high-salt industrial reclaimed water according to claim 1, characterized in that, The machine learning early warning model described in step S5 uses historical detection data, salt concentration data, and production condition data as training sets and is constructed using the random forest algorithm. The model outputs the risk level of microbial exceedance. When the risk level is ≥ medium, the linkage disinfection system starts enhanced disinfection, automatically generates weekly and monthly reports, and supports mobile push notifications.

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