Method for detecting content of microorganisms in water system on line

Abstract

The invention relates to a method for detecting the content of microorganisms in a water system on line, and belongs to the technical field of detection. The method comprises the following steps: directly loading to-be-detected water or loading the to-be-detected water onto a carrier after microorganisms of the to-be-detected water are enriched; transferring the carrier into a closed container with a carbon dioxide sensor, and keeping for a certain time; according to the recorded time and the carbon dioxide release amount, calculating the carbon dioxide release rate of the microorganisms in the to-be-detected water; calculating the total number of microorganisms in the to-be-detected water according to a standard curve of the release rate of carbon dioxide to the total number of microorganisms; and dividing the total number of the microorganisms by the sampling volume to calculate the content of the microorganisms in the to-be-detected water. The method overcomes the defects of traditional detection of microorganisms in water, for example, the detection result has hysteresis quality and cannot reflect the actual condition of the microorganisms in water timely and accurately, so that the bactericide can only be added according to experience or blindly in the actual operation.

Description

Technical Field

[0001] The invention belongs to the field of detection technology, and in particular relates to a method for online detection of microbial content in a water system through changes in carbon dioxide concentration. Background Art

[0002] In many water systems, such as industrial circulating cooling water, reverse osmosis desalted water, and oilfield reinjection water, due to the growth and enrichment of microorganisms, undesirable biofilms grow on surfaces in contact with the water system, such as heat exchangers, water pipes or containers, rock pores, membranes, and membrane pores. These biofilms can cause a decrease in heat exchange efficiency, increase in delivery pressure and energy consumption, a decrease in oilfield production efficiency, and damage to oilfield-related facilities, and a decrease in reverse osmosis membrane efficiency and damage to facilities. The conventional method for controlling and inhibiting excessive biofilm growth is to determine whether to use methods such as adding relevant fungicides to inhibit excessive biofilm growth after detecting the concentration of relevant microorganisms in the water. In such water systems, microorganisms that rely on organic matter as a carbon source and energy source to synthesize the nutrients they need and release carbon dioxide during metabolism, such as heterotrophic bacteria, are the most common, fastest growing and most numerous. Therefore, determining the number of heterotrophic bacteria is an important task in the above system. For example, in the daily monitoring project of circulating cooling water treatment, it is required to measure at least 1 to 2 times a week, and the monitoring index is less than 5×10 5 CFU / mL.

[0003] The traditional method for detecting heterotrophic bacteria concentration is the GB / T 14643.1-2009 plate count method, which is to spread the diluted water sample on a solid culture medium, then culture it under appropriate conditions, and observe the morphology, color and other biochemical indicators of the microorganisms with the naked eye or a microscope to distinguish and calculate the number of microbial colonies. This method has strict requirements on the operator, and each step requires aseptic operation, otherwise counting errors are likely to occur, and the high-temperature sterilization of the culture medium and experimental utensils is time-consuming and laborious. In order to simplify the operating steps, the culture medium filter membrane method has emerged, that is, the water sample is filtered through a filter membrane with a specific pore size, and then the filter membrane is placed on the culture medium for culture, and the number of growing colonies is counted. This method requires the use of pre-made heterotrophic culture medium, which increases the detection cost and does not shorten the detection time, which generally takes 3 to 5 days.

[0004] With the advancement of biotechnology, more and more rapid detection technologies are applied to microbial detection, such as: measuring the ATP content in microbial cells and using bioluminescence reactions for quantification; using specific fluorescent markers to mark microorganisms, and then detecting and analyzing them through flow cytometry; and combining the unique molecules of microorganisms (such as nucleic acids, proteins or metabolites) with the biorecognition elements on the sensor surface to achieve identification and quantification of microorganisms through signal generation and detection. These methods require the use of expensive materials and equipment, and the test results are easily affected by environmental factors, which limits the popularity and application of rapid microbial detection in circulating water.

[0005] After searching, we found that patent CN102618621A provides a quick and easy method for detecting heterotrophic bacteria content in industrial circulating water. The method first filters the water sample to enrich the bacteria in the water sample on the carrier, then hydrolyzes the enriched bacteria, lyses the bacterial film to release protein, and completely hydrolyzes it into amino acids, and then mixes it with the ninhydrin developer to obtain a color solution, measures the absorbance at a wavelength of 562nm, and compares it with the "absorbance-heterotrophic bacteria number" standard curve to obtain the number of heterotrophic bacteria in the water sample. Compared with the traditional national standard plate counting method, the determination method in patent CN102618621A is fast and simple, and the content of heterotrophic bacteria in circulating cooling water can be obtained in a short time; and it is easy to realize instrumentation and automation, and is easy to promote and apply. However, the number of heterotrophic bacteria measured by this method includes not only live bacteria, but also killed bacteria, and the test results may be distorted. Summary of the invention

[0006] The present invention aims to provide a new idea for online rapid detection of microbial content in water. This method overcomes many drawbacks of traditional water microbial detection, such as the lag in the detection results, which cannot timely and accurately reflect the actual status of the microorganisms in the water, resulting in the fact that in actual operation, fungicides can only be added based on experience or blindly. In view of the fact that the dominant microorganisms (such as atypical bacteria) in the target water system release carbon dioxide during their metabolic process, especially during cellular respiration, the concentration of such microorganisms can be reflected by measuring the rate of increase in carbon dioxide concentration. This method has the significant advantages of being fast, accurate and reliable.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for online detection of microbial content in a water system comprises the following steps:

[0009] 1) Load the water to be tested directly or enrich the microorganisms in the water to be tested onto the carrier;

[0010] 2) Transfer the carrier to a sealed container with a carbon dioxide sensor and keep it for a certain period of time; this step records the relationship between the amount of carbon dioxide released by microbial respiration and time;

[0011] 3) According to the time and carbon dioxide release amount recorded in step 2), the rate of carbon dioxide release by the microorganisms in the water to be tested is calculated; in this step, the rate of carbon dioxide release by the microorganisms in the water to be tested is obtained by drawing a time-carbon dioxide release curve;

[0012] 4) Calculate the total number of microorganisms in the water to be tested based on the standard curve of carbon dioxide release rate-total number of microorganisms;

[0013] 5) Divide the total number of microorganisms by the sampling volume to calculate the microbial content in the water to be tested.

[0014] Furthermore, the carrier in step 1) includes but is not limited to filter membranes, filter bags, filter elements, and centrifuge tubes, and the enrichment methods include but are not limited to filtration interception and centrifugal enrichment, and microporous filter membranes of various materials and pore sizes are preferred.

[0015] Furthermore, in the step 2), the sealed container with the carbon dioxide sensor is also provided with a thermostat, and the constant temperature is 20-50°C, preferably 30-40°C.

[0016] Furthermore, in step 2), nutrients and pH stabilizers for maintaining microbial growth are placed in the sealed container according to the detection time of carbon dioxide.

[0017] Furthermore, in step 2), the certain time of maintaining is 20 minutes to 2 hours, preferably 1 hour.

[0018] Furthermore, in the step 4), a standard curve of carbon dioxide release rate-total number of microorganisms is drawn as follows: different volumes of water samples are taken and separated and enriched, and the carrier of enriched microorganisms is placed in a closed container with a carbon dioxide sensor, and the carbon dioxide release amount in a specific time period is detected, and the rate of carbon dioxide release by microorganisms in the water sample (μg / h) is calculated; at the same time, the microbial concentration (CFU / mL) in the water sample is determined by a traditional plate counting method, or other disposable industrial infiltrated agar slides that are easy to handle, and the total number of microorganisms (CFU) in the water sample is obtained from the volume of the water sample and the measured microbial concentration (CFU / mL); a standard curve is drawn with the total number of microorganisms (CFU) in the water sample as the horizontal axis and the corresponding carbon dioxide release rate (μg / h) as the vertical axis to obtain a standard curve of carbon dioxide release rate-total number of microorganisms.

[0019] Furthermore, the plate count method for detecting the content of microorganisms in water samples refers to GB / T 14643.1-2009 "Methods for determination of bacteria and algae in industrial circulating cooling water Part 1: Plate count method for determination of slime-forming bacteria".

[0020] Furthermore, the water system includes but is not limited to the following water systems: circulating cooling water, surface water, geothermal water and drinking water, concentrated water in a reverse osmosis water system, and related water systems in oil and gas production.

[0021] The whole detection process of the method of the present invention is simple and easy to operate, and takes no more than 2 hours. Compared with the traditional plate counting method, this method can obtain the content of microorganisms in water more quickly and accurately, and the whole detection process is easy to realize online automation and industrial application.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The detection method of the present invention is not only simple and rapid, but also accurate and reliable, and requires almost no human intervention. It can obtain the concentration of microorganisms in the target water system that produce carbon dioxide during metabolism in a short time. Based on the above characteristics, it is expected that the present invention has great potential in the detection and intelligent control of water treatment microorganisms in the target water system, including but not limited to the following target water systems: circulating cooling water, surface water, geothermal water and drinking water, concentrated water in reverse osmosis water systems, and related water systems in oil and gas production.

[0024] 2. The present invention not only improves the efficiency of microbial detection, but also provides strong technical support for intelligent control. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The microorganisms in the water to be tested on site are enriched and separated, and then placed in a closed container with a carbon dioxide sensor. The sensor detects the change in the carbon dioxide content produced by the respiration of the microorganisms over time.

[0026] Figure 2 This is the standard curve of carbon dioxide release rate-total number of microorganisms.

[0027] Figure 3 Photo of the Easicult® Combi Total Bacteria Count. DETAILED DESCRIPTION

[0028] The technical solution and effects of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0029] Example 1

[0030] The method for online detection of microbial content in a water system of this embodiment comprises the following steps:

[0031] 1) Take 156 mL of circulating water from a steel plant and enrich the microorganisms in the water sample in a 10 mL centrifuge tube by centrifugation. The centrifuge model is DM0636 and the speed is set to 8000 r / min;

[0032] 2) Quickly transfer the centrifuge tube containing the enriched microorganisms to a sealed container with a carbon dioxide sensor and keep it for 1 hour; this step records the relationship between the amount of carbon dioxide released by microbial respiration and time;

[0033] 3) Calculate the rate at which microorganisms in the water sample release carbon dioxide based on the carbon dioxide release recorded in step 2);

[0034] 4) Calculate the total number of microorganisms in the water sample based on the standard curve of carbon dioxide release rate-total number of microorganisms;

[0035] 4) Divide the total number of microorganisms by the sampling volume to calculate the microbial content in the water sample.

[0036] Example 2

[0037] The method for online detection of microbial content in a water system of this embodiment comprises the following steps:

[0038] 1) Take 3238mL of reverse osmosis water from a resin factory and use membrane filtration to enrich the microorganisms in the water sample on the filter membrane. The filter membrane manufacturer is Jinteng, the pore size is 0.45μm, the diameter is 50mm, and the material is PTFE;

[0039] 2) Quickly transfer the filter membrane enriched with microorganisms to a sealed container with a carbon dioxide sensor and keep it for 1 hour; this step records the relationship between the amount of carbon dioxide released by microbial respiration and time;

[0040] 3) Calculate the rate at which microorganisms in the water sample release carbon dioxide based on the carbon dioxide release recorded in step 2);

[0041] 4) Calculate the total number of microorganisms in the water sample based on the standard curve of carbon dioxide release rate-total number of microorganisms;

[0042] 4) Divide the total number of microorganisms by the sampling volume to calculate the microbial content in the water sample.

[0043] Example 3

[0044] The drawing of the standard curve of carbon dioxide release rate-microorganism total number in this embodiment: take the circulating water of the same period of time on the site of Example 1, and measure the heterotrophic bacteria in the water sample with reference to the plate counting method for measuring microorganisms in GB / T14643.1-2009, so as to determine the total number of heterotrophic bacteria in water samples of different volumes; at the same time, take the water sample of the same period of time on the site of Example 1, and detect the rate of carbon dioxide release by microorganisms in the water sample; draw the standard curve with the rate of carbon dioxide release by microorganisms as the horizontal axis and the number of microorganisms as the vertical axis. The method of detecting microorganisms by plate counting method refers to GB / T14643.1-2009, and the specific operation steps are as follows:

[0045] 1) Preparation of culture medium: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 15.0g agar, add 950mL water, heat the above reagents on an electric stove to dissolve, filter with four layers of medical absorbent gauze while hot, and add hot water to 1000mL, adjust the pH to 7.2±0.2 with sodium hydroxide or hydrochloric acid solution, and divide into 250mL conical flasks. The amount of each bottle should not exceed 2 / 3 of its capacity, plug with cotton, wrap with waste newspaper, and put into an autoclave for sterilization at (121±1)℃ for 15min.

[0046] 2) Preparation of sterile dilution water: Weigh 8.5 g of sodium chloride and dissolve it in 1000 mL of water. Divide the mixture into small test tubes and sterilize them in an autoclave at (121 ± 1) °C for 15 min.

[0047] 3) Sterilization of glassware such as graduated pipettes and culture dishes: Sterilize the wrapped graduated pipettes and culture dishes in an electric oven at (160±1)℃ for 2h.

[0048] 4) Sterilization of the sterile box (room): Place the sterile dilution water, sterile culture medium, sterile graduated pipettes and other experimental supplies used in the experiment into the sterile box (room) and turn on the ultraviolet light for sterilization for 30 minutes.

[0049] 5) Dilution and inoculation of water samples: dilute the circulating water on site by ten-fold dilution method, and inoculate the circulating water samples of different dilutions into sterile culture dishes. Repeat inoculation of 3 dishes for each dilution, 1 mL per dish, and take another set of culture dishes without inoculation of water samples as blanks. Cool the sterilized culture medium to (45±1)℃, pour it into the culture dishes, 15~20 mL per dish, and mix the water sample and culture medium evenly.

[0050] 6) After the culture medium in the culture dish solidifies, place the dish upside down in a biochemical incubator and culture at (29±1)℃ for 72h.

[0051] 7) Observe and count, and find that the content of microorganisms in the water sample is 4.0×10 4CFU / mL. Multiply by the volume of the water sample in turn to get the total number of microorganisms in water samples of different volumes.

[0052] 8) At the same time, water samples were taken from the site of Example 1 at the same time period, and the rate of carbon dioxide release by microorganisms in the water samples was detected according to the steps in Example 1; a standard curve was drawn with the rate of carbon dioxide release by microorganisms as the horizontal axis and the total number of microorganisms as the vertical axis, such as Figure 2 , the standard curve is: y=213.198x 5.75505 , x is the carbon dioxide release rate μg / h, and y is the total number of microorganisms. Figure 2 The specific data of the carbon dioxide release rate and the number of microorganisms in the standard curve are shown in Table 1.

[0053] 9) The method of detecting the total number of microorganisms by plate counting method is only used for the first calibration of the standard curve. The total number of microorganisms of the water samples to be tested in the future only needs to be detected according to the methods of Examples 1 and 2. The rate of carbon dioxide release by microorganisms is detected, and the microbial content in the water sample is calculated based on the standard curve of carbon dioxide release rate-total number of microorganisms.

[0054] Table 1 Standard curve data of carbon dioxide release rate-microorganism count

[0055]

[0056] Example 4

[0057] Water samples were randomly collected from different sites and the following procedures were followed:

[0058] 1) Filter the water sample with a membrane filter to enrich the bacteria in the water sample on the carrier. The pore size of the membrane filter is 0.22 μm and the material is PES.

[0059] 2) Rapidly transfer the bacteria-enriched carrier to a sealed container with a carbon dioxide sensor and keep it for 1 hour; this step records the relationship between the amount of carbon dioxide released by bacterial respiration and time;

[0060] 3) Calculate the number of microorganisms in the water sample based on the carbon dioxide release rate combined with the standard curve of carbon dioxide release rate-total number of microorganisms.

[0061] 4) The total number of microorganisms obtained was compared with the Easicult® Combi bacterial count plate to verify the accuracy of the detection method. The obtained microbial content is shown in Table 2. Figure 3 The figure is a photo of the Easicult® Combi bacterial count plate method. The numbers 1 and 2 in Table 2 are the corresponding data of Example 1 and Example 2 respectively.

[0062] Table 2 Comparison of total microbial counts detected by carbon dioxide method and Easicult® Combi total bacterial count method

[0063]

[0064] According to Table 2, the total amount of microorganisms obtained by detecting the carbon dioxide concentration is basically consistent with the result obtained by the commercially available Easicult® Combi method of detecting the total number of bacteria. Therefore, the content of microorganisms in the system can be quickly and conveniently monitored by online detection of changes in the concentration of carbon dioxide in the circulating water.

[0065] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for online detection of microbial content in a water system, characterized in that: The method comprises the following steps: 1) Load the water to be tested directly or enrich the microorganisms in the water to be tested onto the carrier; 2) Transfer the carrier to a sealed container with a carbon dioxide sensor and keep it for a certain period of time; this step records the relationship between the amount of carbon dioxide released by microbial respiration and time; 3) According to the time and carbon dioxide release amount recorded in step 2), the rate of carbon dioxide release by the microorganisms in the water to be tested is calculated; in this step, the rate of carbon dioxide release by the microorganisms in the water to be tested is obtained by drawing a time-carbon dioxide release curve; 4) Calculate the total number of microorganisms in the water to be tested based on the standard curve of carbon dioxide release rate-total number of microorganisms; 5) Divide the total number of microorganisms by the sampling volume to calculate the microbial content in the water to be tested.

2. The method for online detection of microbial content in a water system according to claim 1, characterized in that: The carrier in step 1) includes but is not limited to filter membranes, filter bags, filter elements, and centrifuge tubes, and the enrichment method includes but is not limited to filtration interception and centrifugal enrichment. Microporous filter membranes of various materials and pore sizes are preferred.

3. The method for online detection of microbial content in a water system according to claim 1, characterized in that: In the step 2), the sealed container with the carbon dioxide sensor is also provided with a constant temperature device, and the constant temperature is 20-50°C, preferably 30-40°C.

4. The method for online detection of microbial content in a water system according to claim 1, characterized in that: In the step 2), nutrients and pH stabilizers for maintaining microbial growth are placed in the sealed container according to the detection time of carbon dioxide.

5. The method for online detection of microbial content in a water system according to claim 1, characterized in that: The step 2) is maintained for a certain time of 20 minutes to 2 hours, preferably 1 hour.

6. The method for online detection of microbial content in a water system according to claim 1, characterized in that: The standard curve of carbon dioxide release rate-total number of microorganisms in step 4) is drawn as follows: different volumes of water samples are taken and separated and enriched, and the carrier of enriched microorganisms is placed in a closed container with a carbon dioxide sensor, and the carbon dioxide release amount in a specific time period is detected, and the carbon dioxide release rate of microorganisms in the water sample is calculated in μg / h; at the same time, the microbial concentration CFU / mL in the water sample is determined by the traditional plate counting method, or other easy-to-handle disposable industrial infiltrated agar slides are used to determine the microbial concentration CFU / mL in the water sample, and the total number of microorganisms CFU in the water sample is obtained from the water sample volume and the measured microbial concentration CFU / mL; a standard curve is drawn with the total number of microorganisms CFU in the water sample as the horizontal axis and the corresponding carbon dioxide release rate μg / h as the vertical axis to obtain a standard curve of carbon dioxide release rate-total number of microorganisms.

7. The method for online detection of microbial content in a water system according to claim 6, characterized in that: The plate count method for detecting the content of microorganisms in water samples refers to GB / T 14643.1-2009 "Methods for determination of bacteria and algae in industrial circulating cooling water Part 1: Plate count method for determination of slime-forming bacteria".

8. The method for online detection of microbial content in a water system according to claim 1, characterized in that: The water system includes but is not limited to the following water systems: circulating cooling water, surface water, geothermal water and drinking water, concentrated water in the reverse osmosis water system, and related water systems in oil and gas production.

Images