Culture system suitable for detecting sulfate reducing bacteria in complex field environment, application and detection method

Abstract

The invention belongs to the technical field of bacterium detection, and particularly relates to a culture system suitable for detecting sulfate reducing bacteria (SRB) in a complex field environment, application and a detection method. The culture system is prepared by preparing an SRB detection culture medium, introducing atmosphere gas into the culture medium and performing high-temperature sterilization; wherein the atmosphere gas is a mixture of CO2 and H2. The culture system is applied to detection of sulfate reducing bacteria (SRB) in a complex field environment. The rapid SRB detection method has the characteristics of rapidness, low detection limit and simplicity and convenience in operation.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacteria detection, and in particular relates to a culture system, application and detection method suitable for detecting sulfate-reducing bacteria (SRB) in complex on-site environments. Background Art

[0002] Sulfate-reducing bacteria (SRB) are a group of prokaryotic microorganisms in ecosystems. SRB are widely found in soil, seawater, river water, underground pipelines, oil and gas wells, and other places. SRB is a typical corrosive bacterium that can use organic matter in the environment to reduce sulfate or other oxidized sulfides during its metabolic process. In addition to producing carbon dioxide and water, this metabolic activity also produces hydrogen sulfide. The production of hydrogen sulfide can cause serious chemical corrosion hazards to contact metals, especially carbon steel, and is one of the problems that need to be overcome in marine engineering. Under special environmental conditions, SRB can also directly use metals or polarized electrodes as electron donors to reduce sulfate, posing a research hazard to marine engineering facilities.

[0003] In the detection of sulfate-reducing bacteria, the current methods involved have their own advantages and disadvantages. The detection method based on characteristic compounds is currently the most commonly used and most effective field experimental method. However, the detection limit of the APS enzyme rapid detection method on the market can only reach 10 3 Given the widespread presence of SRB in various environments and their serious harm to engineering facilities, it is extremely important to detect and monitor their concentration in the environment. Summary of the Invention

[0004] Different testing technologies have different advantages and disadvantages. In response to the above problems and the shortcomings of current technologies, the present invention aims to provide a culture system, application and detection method suitable for detecting sulfate-reducing bacteria (SRB) in complex field environments.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A culture system suitable for detecting sulfate-reducing bacteria in complex field environments is provided. The culture system is prepared by configuring an SRB detection culture medium, introducing an atmospheric gas into the culture medium, and sterilizing the culture medium at high temperature. The atmospheric gas is a mixture of CO2 and H2.

[0007] The culture medium is introduced with a mixed gas of CO2 and H2 until the system is completely filled with atmospheric CO2 and H2, and then sterilized at high temperature to obtain a culture system; wherein the volume ratio of CO2 to H2 is 8:2.

[0008] The SRB detection culture medium is composed of 50mM Na2SO4, 30mM NaC3H5O3, 8.0mMMgCl2, 20mM NH4Cl, 2.2mM K2HPO4 / KH2PO4 (pH7.2), 0.6mM CaCl2, 10mL vitamin solution, 12.5mL trace mineral solution, 30Mm PIPES buffer, 0.06mM resazurin, and 10mM NaOH (pH7.2) per liter of seawater.

[0009] The vitamins are prepared according to the following ratio per liter of distilled water: 2 mg biotin; 2 mg folic acid; 10 mg pyridoxine hydrochloride; 5 mg thiamine hydrochloride; 5 mg riboflavin; 5 mg niacin; 5 mg DL-calcium pantothenate; 0.1 mg vitamin B12; 5 mg p-aminobenzoic acid; 5 mg lipoic acid; and 200 mg choline chloride. The solution is stored at 4° C. under N2 in the dark.

[0010] The trace mineral solution is prepared according to the following ratio per liter of distilled water: 12.8 g aminotriacetic acid (adjusted to pH 6.5 with NaOH); 1 g Fe2Cl.4H2O; 0.5 g MnCl2.4H2O; 0.3 g CoCl2.6H2O; 0.2 g zinc chloride; 50 mg sodium molybdate dihydrate; and 20 mg H3BO3.

[0011] An application of the culture system is the application of the culture system to detect sulfate-reducing bacteria (SRB) in a complex field environment.

[0012] A method for detecting sulfate-reducing bacteria in complex field environments:

[0013] S1. Process samples;

[0014] S2. configuring the culture system according to claim 1;

[0015] S3, dilute the sample obtained from S1 and then culture it in the S2 culture system;

[0016] S4. Culture the sample at room temperature and detect sulfate-reducing bacteria.

[0017] The S3 is to perform a concentration gradient dilution on the bacteria in step S1, add the diluted bacteria into the S2 culture system and place the bacteria in a constant temperature incubator at 25-35°C for constant temperature culture.

[0018] The step S1 sample processing:

[0019] (1) For liquids: Take a 1 L volume of water sample to be tested and filter it through a 0.22 μm filter membrane. After removing the filter membrane, rinse it with 0.1 M PBS buffer and centrifuge it at 14000 g for 15 minutes. Finally, concentrate the water sample to 1.5 mL and collect it in a centrifuge tube. Store it at 4°C.

[0020] (2) For sediment samples, remove obvious impurities, grind with a mortar, add 978 μL sodium phosphate buffer and 122 μL MT Buffer to the sample, place the centrifuge tube in a shaker, shake at a speed of 6.0 for 40 seconds, centrifuge at 14000g for 15 minutes, and store at 4°C.

[0021] The concentration gradient dilution method is specifically as follows: fill the test bottles with 9mL PBS solution and arrange them in a group, and number them in sequence. Use a sterile syringe to take 1ml of the sample after step S1 treatment, inject it into bottle No. 1 for dilution, shake it thoroughly, and set aside. Use another sterile syringe to extract 1ml of the water sample from bottle No. 1 and shake it thoroughly, inject it into bottle No. 2 for dilution and shake it thoroughly, and set aside. Use another sterile syringe to extract 1ml of the water sample from bottle No. 2 and shake it thoroughly, inject it into bottle No. 3 for dilution and shake it thoroughly, and set aside. Repeat the dilution process until the last bottle is reached and set aside. The bacterial culture method is specifically as follows: add the diluted bacterial solution to the S2 culture system at a 2% inoculation rate. Place all the above test bottles in a constant temperature incubator (29±1°C) for culture.

[0022] The method measures sulfate consumption in a sample over a period of time and establishes a relationship curve between the consumption and bacterial concentration, thereby enabling rapid detection of sulfate-reducing bacteria concentration in complex field environments.

[0023] The beneficial effects of the present invention are:

[0024] The rapid detection method of SRB of the present invention has the characteristics of rapidity, low detection limit and simple operation.

[0025] (1) Compared with the traditional extinction dilution method, the present invention greatly shortens the detection time to 7 days.

[0026] (2) Compared with the traditional extinction dilution method, the detection sensitivity of the present invention is improved by 10 times.

[0027] (3) The present invention is simple to operate, convenient and feasible, and can be adapted to the rapid detection of SRB in complex field environments. DETAILED DESCRIPTION

[0028] The specific embodiments of the present invention are further described below with reference to examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.

[0029] The present invention designs a novel culture system that significantly shortens the preparation time of traditional MPN by an order of magnitude. By measuring sulfate consumption over time at different SRB concentrations, a curve is established between bacterial concentration and sulfate consumption, enabling rapid SRB detection. This invention applies laboratory techniques to SRB detection in real-world environments, minimizing the influence of environmental factors and achieving rapid SRB detection.

[0030] Example:

[0031] S1. Extract 1 L of an environmental sample (which can be oilfield discharge fluid, sewage, seawater, or other aqueous solution; in this example, oilfield discharge fluid) and filter it using a 0.22 μm filter membrane to concentrate the microorganisms in the environmental sample into the filter membrane. Transfer the bacteria-enriched filter membrane to a sterile PBS solution and sonicate to suspend the bacteria in 10 mL of PBS solution.

[0032] S2. Prepare SRB detection culture medium and introduce a mixed gas of CO2 and H2 into the culture medium until the system is completely filled with atmospheric CO2 and H2, and then sterilize at high temperature (121°C, 30 minutes) to obtain the culture system; wherein, the volume ratio of CO2 to H2 is 8:2.

[0033] The SRB detection culture medium is composed of 50mM Na2SO4, 30mM NaC3H5O3, 8.0mMMgCl2, 20mM NH4Cl, 2.2mM K2HPO4 / KH2PO4 (pH7.2), 0.6mM CaCl2, 10mL vitamin solution, 12.5mL trace mineral solution, 30Mm PIPES buffer, 0.06mM resazurin, and 10mM NaOH (pH7.2) per liter of seawater.

[0034] The vitamins are prepared according to the following ratio per liter of distilled water: 2 mg biotin; 2 mg folic acid; 10 mg pyridoxine hydrochloride; 5 mg thiamine hydrochloride; 5 mg riboflavin; 5 mg niacin; 5 mg DL-calcium pantothenate; 0.1 mg vitamin B12; 5 mg p-aminobenzoic acid; 5 mg lipoic acid; and 200 mg choline chloride. The solution is stored at 4° C. under N2 in the dark.

[0035] The trace mineral solution is prepared according to the following ratio per liter of distilled water: 12.8 g aminotriacetic acid (adjusted to pH 6.5 with NaOH); 1 g Fe2Cl.4H2O; 0.5 g MnCl2.4H2O; 0.3 g CoCl2.6H2O; 0.2 g zinc chloride; 50 mg sodium molybdate dihydrate; and 20 mg H3BO3.

[0036] S3. Use a sterile syringe to draw 1 mL of PBS solution containing dissolved bacteria from the environmental sample and inject it into a sealed bottle containing 99 mL of the prepared culture system for culture.

[0037] S4. After culturing at room temperature for different days, SRB were counted (see Table 1).

[0038] Comparative Example 1:

[0039] S1. Extract 1 L of aqueous solution from an actual sewage environment (the same sample as described in Example 1) and filter it using a 0.22 μm filter membrane to enrich the microbial bacteria in the environmental sample into the filter membrane. Transfer the filter membrane enriched with bacteria to a sterile PBS solution and sonicate to suspend the bacteria in 10 mL of PBS solution.

[0040] S2. Prepare SRB detection culture medium and introduce pure CO2 (greater than 99%) gas into the culture medium until the system is completely filled with pure CO2 mixed gas, and then sterilize at 121°C to obtain the culture system.

[0041] The SRB detection culture medium is composed of 50mM Na2SO4, 30mM NaC3H5O3, 8.0mMMgCl2, 20mM NH4Cl, 2.2mM K2HPO4 / KH2PO4 (pH7.2), 0.6mM CaCl2, 10mL vitamin solution, 12.5mL trace mineral solution, 30Mm PIPES buffer, 0.06mM resazurin, and 10mM NaOH (pH7.2) per liter of seawater.

[0042] The vitamins are prepared according to the following ratio per liter of distilled water: 2 mg biotin; 2 mg folic acid; 10 mg pyridoxine hydrochloride; 5 mg thiamine hydrochloride; 5 mg riboflavin; 5 mg niacin; 5 mg DL-calcium pantothenate; 0.1 mg vitamin B12; 5 mg p-aminobenzoic acid; 5 mg lipoic acid; and 200 mg choline chloride. The solution is stored at 4° C. under N2 in the dark.

[0043] The trace mineral solution is prepared according to the following ratio per liter of distilled water: 12.8 g aminotriacetic acid (adjusted to pH 6.5 with NaOH); 1 g Fe2Cl.4H2O; 0.5 g MnCl2.4H2O; 0.3 g CoCl2.6H2O; 0.2 g zinc chloride; 50 mg sodium molybdate dihydrate; and 20 mg H3BO3.

[0044] S3. Use a sterile syringe to draw 1 mL of PBS solution containing dissolved bacteria from the environmental sample and inject it into a sealed bottle containing 99 mL of the prepared culture system for culture.

[0045] S4. After culturing at room temperature for different days, SRB were counted (see Table 1).

[0046] Comparative Example 2:

[0047] According to the national standard "Determination of bacteria and algae in industrial circulating cooling water Part 5: Determination of sulfate-reducing bacteria MPN method" GB / T 14643.5-2009, prepare the bacterial culture medium:

[0048] S1. Extract 1 L of aqueous solution from an actual sewage environment (the same sample as described in Example 1) and filter it using a 0.22 μm filter membrane to enrich the microbial bacteria in the environmental sample into the filter membrane. Transfer the filter membrane enriched with bacteria to a sterile PBS solution and sonicate to suspend the bacteria in 10 mL of PBS solution.

[0049] S2. Prepare SRB detection medium (0.5g potassium hydrogen phosphate, 1.0g ammonium chloride, 0.5g sodium sulfate, 0.1g calcium chloride, 2.0g magnesium sulfate, 3.5g sodium lactate, 1.0g yeast extract), dissolve the above reagents in 1000mL of water, adjust the pH to 7.2±0.2 with sodium hydroxide solution or hydrochloric acid solution, and divide the mixture into 500mL, graduated Erlenmeyer flasks, with each bottle not exceeding 350mL. Cover the bottle mouth with cotton and wrap it with kraft paper. Sterilize it in a steam pressure sterilizer at 121+1℃ for 15min.

[0050] Ammonium ferrous sulfate solution: On the day the culture medium is used, weigh 1.2 g of ammonium ferrous sulfate and spread it evenly in a sterile box (room) at a distance of 30 cm from the ultraviolet lamp for sterilization for 30 minutes. Under aseptic operation, dissolve the ammonium ferrous sulfate in the sterile water prepared in advance and mix well.

[0051] Vitamin C solution: On the day the culture medium is used, weigh 0.4g of vitamin C and spread it evenly in a sterile box (room) at a distance of 30cm from the UV lamp for sterilization for 30 minutes. Under aseptic operation, dissolve the vitamin C in the prepared sterile water and mix thoroughly.

[0052] At the same time, a SRB test bottle purchased from the market was used as control example 3 for comparison to verify the feasibility of the method of the present invention.

[0053] Table 1 Test results of environmental samples using different detection methods

[0054] Number of days Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3: SRB test bottle purchased from the market 7 <![CDATA[3.5×10 3 ]]> <![CDATA[1.5×10 2 ]]> <![CDATA[1.5×10 2 ]]> 0 21 <![CDATA[3.5×10 3 ]]> <![CDATA[1.5×10 2 ]]> <![CDATA[1.5×10 2 ]]> 0

Claims

1. A culture system suitable for detecting sulfate-reducing bacteria in complex field environments, characterized by: The SRB detection culture medium is prepared, atmospheric gas is introduced into the culture medium, and high-temperature sterilization is performed to form a culture system; wherein the atmospheric gas is a mixture of CO2 and H2.

2. The culture system for detecting sulfate-reducing bacteria in complex field environments according to claim 1, characterized in that: The culture medium is introduced with a mixed gas of CO2 and H2 until the system is completely filled with atmospheric CO2 and H2, and then sterilized at high temperature to obtain a culture system; wherein the volume ratio of CO2 to H2 is 8:

2.

3. The culture system for detecting sulfate-reducing bacteria in complex field environments according to claim 1 or 2, characterized in that: The SRB detection medium consists of 50mM Na2SO4, 30mM NaC3H5O3, 8.0mM MgCl2, 20mMNH4Cl, 2.2mM K2HPO4 / KH2PO4 (pH7.2), 0.6mM CaCl2, 10mL vitamin solution, 12.5mL trace mineral solution, 30Mm PIPES buffer, 0.06mM resazurin, and 10mM NaOH (pH7.2) per liter of seawater.

4. The culture system suitable for detecting sulfate-reducing bacteria in complex field environments according to claim 3, characterized in that: The vitamins are 2mg biotin and 2mg folic acid per liter of distilled water; Prepare a mixture of 10 mg pyridoxine hydrochloride, 5 mg thiamine hydrochloride, 5 mg riboflavin, 5 mg niacin, 5 mg DL-calcium pantothenate, 0.1 mg vitamin B12, 5 mg p-aminobenzoic acid, 5 mg lipoic acid, and 200 mg choline chloride, and store the solution at 4°C under nitrogen in the dark. The trace mineral solution is as follows: 12.8 g aminotriacetic acid (adjusted to pH 6.5 with NaOH) per liter of distilled water; 1 g Fe2Cl.4H2O; The ratio of preparation is 0.5g MnCl2.4H2O; 0.3g CoCl2.6H2O; 0.2g zinc chloride; 50mg sodium molybdate dihydrate; 20mg H3BO3.

5. A use of the culture system according to claim 1, characterized in that: The culture system is used to detect sulfate-reducing bacteria (SRB) in complex field environments.

6. A method for detecting sulfate-reducing bacteria in a complex field environment, characterized by: S1. Process samples; S2. configuring the culture system according to claim 1; S3, dilute the sample obtained from S1 and then culture it in the S2 culture system; S4. Culture the sample at room temperature and detect sulfate-reducing bacteria.

7. The method according to claim 6, characterized in that: The S3 is to perform a concentration gradient dilution on the bacteria in step S1, add the diluted bacteria into the S2 culture system and place the bacteria in a constant temperature incubator at 25-35°C for constant temperature culture.

8. The method according to claim 6, wherein: The step S1 sample processing: (1) For liquids: Take 1 L of the water sample to be tested and filter it through a 0.22 μm filter membrane. Remove the filter membrane and rinse it with 0.1 M PBS buffer. Centrifuge it at 14,000 g for 15 minutes. Concentrate the water sample to 1.5 mL and collect it in a centrifuge tube. Store it at 4°C. (2) For sediment samples, remove obvious impurities, grind with a mortar, add 978 μL sodium phosphate buffer and 122 μL MT Buffer to the sample, place the centrifuge tube in a shaker, shake at a speed of 6.0 for 40 seconds, centrifuge at 14000g for 15 minutes, and store at 4°C.