Manganese-oxidizing bacterial culture medium suitable for low-carbon source environment in water supply network and application

Abstract

This invention discloses a culture medium for manganese-oxidizing bacteria suitable for low-carbon source environments in water supply networks and its application. The culture medium is prepared by mixing ferric ammonium citrate, manganese sulfate, ammonium sulfate, sodium chloride, magnesium sulfate, dipotassium hydrogen phosphate, and sodium nitrate, followed by high-temperature and high-pressure sterilization to obtain a culture solution. Manganese sulfate is added to the culture solution to obtain the manganese-oxidizing bacteria culture medium. Manganese-oxidizing bacteria are screened using this culture medium. Cast iron pipe scale is subcultured in the medium to enrich the manganese-oxidizing bacteria culture solution. The enriched manganese-oxidizing bacteria culture solution is diluted and spread onto agar solid medium, and incubated at a constant temperature to obtain manganese-oxidizing bacteria colonies. This invention solves the problem of altered bacterial physiological metabolism caused by high concentrations of added manganese ions, effectively improving bacterial activity and manganese oxidation capacity.

Description

Technical Field

[0001] This invention relates to the field of microbial culture in water supply networks, specifically to a culture medium composition and specific screening and cultivation method suitable for screening highly efficient manganese-oxidizing bacteria in low-carbon source environments of water supply networks. Background Technology

[0002] In the water supply and distribution process, the "yellow water" problem has always been a significant challenge to water supply safety, and the accumulation of manganese in the water supply network is one of the key contributing factors. When the amount of disinfectant remaining in the network is insufficient, the dissolved divalent manganese ions (Mn(II)) in the water easily become the "metabolic substrate" for manganese-oxidizing bacteria. These bacteria oxidize the divalent manganese ions into insoluble manganese oxides, which gradually deposit on the inner wall of the network. Once there are drastic changes in hydraulic conditions (such as the restoration of water supply after network maintenance, the start and stop of water pumps, etc.) or fluctuations in water quality parameters (changes in pH value and dissolved oxygen), a large amount of manganese oxide deposits on the pipe wall will fall into the water, causing a sharp increase in water turbidity. The tap water flowing out will appear as "yellow water" or even "black water," seriously threatening the safety of drinking water quality.

[0003] Currently, the culture media used for screening manganese-oxidizing bacteria from water supply networks have several limitations, including poor specificity and low manganese oxidation efficiency of the screened bacteria. Traditional culture media mostly use general nutrient formulas with high carbon sources, which cannot accurately adapt to the specific nutritional needs and growth environment of manganese-oxidizing bacteria in water supply networks, resulting in low screening efficiency or even failure to screen bacteria.

[0004] Existing culture media for manganese-oxidizing bacteria generally use high concentrations of organic carbon sources (such as peptone, yeast extract, etc.). Although this can promote rapid bacterial growth, it can easily lead to the proliferation of a large number of contaminating bacteria, thereby inhibiting the growth of the target manganese-oxidizing bacteria and reducing screening efficiency and accuracy.

[0005] The concentration of manganese ions added to existing manganese-oxidizing bacterial culture media is often high (commonly ranging from 50 to 500 mg / L or even higher), which far exceeds the actual concentration of manganese ions present in water supply networks (the Mn standard in the "Standards for Drinking Water Quality" (GB 5749-2022) specifies). 2+ Concentration <0.1 mg / L). High concentrations of manganese can alter the physiological and metabolic behavior of bacteria, and the strains screened may not be manganese-oxidizing bacteria found in the water supply network. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for preparing a culture medium for manganese-oxidizing bacteria in a low-carbon source environment of a water supply network. This method effectively screens out the culture medium components for highly efficient manganese-oxidizing bacteria from the low-carbon source environment of the water supply network, precisely adapts to the special nutritional needs and growth environment of manganese-oxidizing bacteria in the water supply network, and improves screening efficiency and accuracy.

[0007] The second objective of this invention is to provide a method for screening manganese-oxidizing bacteria based on a culture medium containing manganese-oxidizing bacteria, thereby solving the problem that high concentrations of added manganese ions alter the physiological metabolism of bacteria and effectively improving bacterial activity and manganese oxidation capacity.

[0008] The present invention is achieved through the following technical solution.

[0009] According to one aspect of the present invention, a method for preparing a culture medium for manganese-oxidizing bacteria suitable for low-carbon source environments in water supply networks is provided, comprising: a. Prepare the culture medium according to the following mass ratio of raw materials: 2.55~2.65 parts ferric ammonium citrate, 1.95~2.05 parts MnSO4·H2O, 490~510 parts (NH4)2SO4, 190~210 parts NaCl, 250~260 parts MgSO4·7H2O, 490~510 parts K2HPO4 and 490~510 parts NaNO3; b. Adjust the pH of the culture medium to neutral, and then sterilize it under high temperature and high pressure to obtain the culture solution; c. After the culture medium has cooled, add MnSO4·H2O through a microporous filter membrane to obtain a culture medium for manganese-oxidizing bacteria.

[0010] Preferably, the pH of the culture medium is adjusted to 6.8-7.2 using HCl and NaOH solutions.

[0011] Preferably, sterilization is performed at a high temperature of 120~130℃ for 30~60 minutes.

[0012] Preferably, the culture medium is cooled to 25-35 °C, and 4.9-5.1 mL of 300-500 mg / L MnSO4·H2O is added through a 0.22 µm microporous membrane on a UV-sterilized work surface.

[0013] In another aspect, the present invention provides a culture medium for manganese-oxidizing bacteria prepared by the method described above, suitable for use in low-carbon source environments of water supply networks.

[0014] In another aspect, the present invention provides a method for screening manganese-oxidizing bacteria using a manganese-oxidizing bacteria culture medium, comprising: a. Scrape 1-5 parts of scale from cast iron water supply pipe network, place them in 200-300 parts of liquid manganese oxidizing bacteria culture medium, and carry out subculture under constant temperature shaking to obtain manganese oxidizing bacteria suspension; b. Add 5-10 parts of bacterial suspension culture medium to 200-300 parts of liquid manganese oxidizing bacteria culture medium, and repeat the operation for several culture cycles to obtain enriched manganese oxidizing bacteria culture. c. Dilute the enriched culture of manganese-oxidizing bacteria and spread it on a solid culture medium containing agar, and incubate at a constant temperature until bacterial colonies appear; d. Use LBB indicator to screen for bacterial colonies with manganese oxidizing properties; bacterial colonies that turn blue are manganese oxidizing bacterial colonies.

[0015] Preferably, the scale in the cast iron water supply network is placed in a liquid culture medium and cultured under constant temperature shaking at a temperature of 20~30 ℃ and a rotation speed of 100~200 r / min, with a cycle of 4~6 days.

[0016] The preferred method for preparing agar-containing solid culture medium is as follows: Add 15-20 g / L agar powder to the liquid culture medium, shake well, and slowly pour it into the sterilized plate to a thickness of 2-3 mm. After cooling and solidification, seal the plate and store it upside down in a constant temperature incubator.

[0017] Preferably, the bacteria are incubated at a constant temperature of 20-30 ℃ in an incubator until colonies of different shapes and colors appear.

[0018] Preferably, the bacterial culture is diluted using a ratio method of 10:10. 1 ~10 6 Diluted bacterial solutions of 100-200 µL were spread onto solid agar plates on a sterile table using sterilized pipette tips and spreaders.

[0019] Preferably, the LBB colorimetric method uses LBB indicator, and the preparation method is as follows: Take 0.04~0.06 g of LBB powder by mass ratio, dissolve it in 0.25~0.30 mL of 40~50 mmol / L glacial acetic acid solution, add ultrapure water to make up to 100 mL, and store at 4 ℃ protected from light.

[0020] The present invention, by adopting the above technical solution, has the following beneficial effects: 1. This invention uses ferric ammonium citrate as the sole carbon source in the culture medium, which is closer to the low-carbon environment in the water supply network. This avoids the situation where some culture media contain too much readily available carbon source, leading to the proliferation of miscellaneous bacteria and inhibiting the growth of manganese-oxidizing bacteria. This allows for better screening of manganese-oxidizing bacteria in low-carbon environments such as water supply networks.

[0021] 2. This invention isolates target bacteria from scale in cast iron water supply pipes. Through repeated enrichment culture and dilution, the activity and manganese oxidation capacity of the bacteria can be effectively improved, with a manganese oxidation rate of up to 99.67%. This is beneficial for studying the manganese oxidation mechanism of manganese-oxidizing bacteria in water supply networks.

[0022] 3. Using a triangular glass rod heated by an alcohol lamp to spread the bacterial solution can avoid damaging the surface of the plate and further maintain the integrity of the culture medium.

[0023] 4. Inverted culture can avoid the generation of water vapor, thus preventing water vapor from affecting the observation of bacterial morphology and quantity.

[0024] 5. Combine serial dilution with plate plating to isolate colonies, controlling the dilution factor to 10. 1 ~10 6 This can prevent the problem of insufficient colony counts to provide enough research subjects for subsequent studies, as well as the problem of excessive colony counts leading to mutual compression, overlap, spread, and difficulty in distinguishing between colonies, which affects bacterial growth and metabolism. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a plate image of manganese-oxidizing bacteria screened from a water supply network in an embodiment of the present invention; Figure 2 Different Mg obtained in this invention 2+ Growth curves of manganese-oxidizing bacteria in water supply networks under specific concentration conditions; Figure 3 Different Fe obtained in this invention 2+ Oxidation characteristics curves of manganese-oxidizing bacteria in water supply networks under certain concentration conditions. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0027] This invention provides a method for preparing a culture medium for manganese-oxidizing bacteria suitable for low-carbon source environments in water supply networks, comprising the following steps: Step 1: Prepare the culture medium raw materials according to the following mass ratio: 2.55~2.65 parts ferric ammonium citrate, 1.95~2.05 parts MnSO4·H2O, 490~510 parts (NH4)2SO4, 190~210 parts NaCl, 250~260 parts MgSO4·7H2O, 490~510 parts K2HPO4 and 490~510 parts NaNO3.

[0028] Step 2: Weigh the above components using an electronic balance and add them to an Erlenmeyer flask. Dilute to the mark with ultrapure water, then adjust the pH to 6.8-7.2 with NaOH and HCl solutions. After high-temperature and high-pressure sterilization, the culture medium is obtained.

[0029] Step 3: After sealing the culture medium with sealing film and kraft paper, sterilize it at 120~130 ℃ for 30~60 min. After sterilization, wait for the culture medium to cool to 25~35 ℃, and add 4.90~5.10 mL of 300~500 mg / L MnSO4·H2O through a 0.22 µm microporous membrane on a sterile table to obtain manganese oxidizing bacteria culture medium.

[0030] In this invention, ferric ammonium citrate, as the sole carbon source in the culture medium, more closely resembles the low-carbon environment within the water supply network. This avoids the situation where some culture media contain excessive readily available carbon sources, leading to the proliferation of miscellaneous bacteria and inhibiting the growth of manganese-oxidizing bacteria. Furthermore, ammonium nitrogen and nitrate nitrogen, such as ferric ammonium citrate, (NH4)2SO4, and NaNO3, serve as abundant nitrogen sources that can meet the growth requirements of different types of manganese-oxidizing bacteria, facilitating the screening of various types of manganese-oxidizing bacteria.

[0031] The preparation of the manganese-oxidizing bacterial culture medium of the present invention will be further illustrated below through different embodiments.

[0032] Example 1 (1) Prepare the culture medium raw materials according to the following mass ratio: 2.60 parts ferric ammonium citrate, 2.0 parts MnSO4·H2O, 500 parts (NH4)2SO4, 200 parts NaCl, 255 parts MgSO4·7H2O, 500 parts K2HPO4 and 500 parts NaNO3.

[0033] (2) Weigh the above components using an electronic balance and add them to an Erlenmeyer flask. Make up to the mark with ultrapure water, then adjust the pH to 7.0 with NaOH and HCl solution. After high temperature and high pressure sterilization, the culture medium is obtained.

[0034] (3) After sealing the culture medium with sealing film and kraft paper, sterilize it at 125℃ for 40 min. After sterilization, wait for the culture medium to cool to 30℃, and add 5.0 mL of 400 mg / L MnSO4·H2O through a 0.22 µm microporous filter membrane on a sterile platform to obtain manganese oxidizing bacteria culture medium.

[0035] Example 2 (1) Prepare the culture medium raw materials according to the following mass ratio: 2.65 parts ferric ammonium citrate, 1.95 parts MnSO4·H2O, 490 parts (NH4)2SO4, 190 parts NaCl, 260 parts MgSO4·7H2O, 490 parts K2HPO4 and 510 parts NaNO3.

[0036] (2) Weigh the above components using an electronic balance and add them to an Erlenmeyer flask. Make up to the mark with ultrapure water, then adjust the pH to 6.8 with NaOH and HCl solution. After high temperature and high pressure sterilization, the culture medium is obtained.

[0037] (3) After sealing the culture medium with sealing film and kraft paper, sterilize it at 130 °C for 30 min. After sterilization, wait for the culture medium to cool to 25 °C, and add 5.10 mL of 500 mg / L MnSO4·H2O through a 0.22 µm microporous filter membrane on a sterile platform to obtain manganese oxidizing bacteria culture medium.

[0038] Example 3 (1) Prepare the culture medium raw materials according to the following mass ratio: 2.50 parts ferric ammonium citrate, 2.05 parts MnSO4·H2O, 510 parts (NH4)2SO4, 210 parts NaCl, 250~260 parts MgSO4·7H2O, 510 parts K2HPO4 and 490 parts NaNO3.

[0039] (2) Weigh the above components using an electronic balance and add them to an Erlenmeyer flask. Make up to the mark with ultrapure water, then adjust the pH to 7.2 with NaOH and HCl solution. After high temperature and high pressure sterilization, the culture medium is obtained.

[0040] (3) After sealing the culture medium with sealing film and kraft paper, sterilize it at 120 °C for 60 min. After sterilization, wait for the culture medium to cool to 35 °C, and add 4.90 mL of 300 mg / L MnSO4·H2O through a 0.22 µm microporous filter membrane on a sterile table to obtain manganese oxidizing bacteria culture medium.

[0041] Furthermore, the present invention also provides a method for screening manganese-oxidizing bacteria from a manganese-oxidizing bacteria culture medium, comprising the following steps: Bacterial enrichment culture was carried out using liquid culture medium, and then the bacteria were diluted and spread on solid culture medium. After the bacterial colonies were cultured, LBB chromogenic reagent was used to screen and obtain manganese oxidizing bacterial colonies.

[0042] Step 1: Scrape 1-5 g of scale from cast iron water supply pipes and add it to 250 mL of liquid culture medium. Seal the container with sealing film and place it in a constant temperature shaking incubator. Under the conditions of 20-30 ℃ and 100-200 r / min, carry out subculture. Subculture is carried out in cycles of 4-6 days. After 4-6 days, a suspension of manganese oxidizing bacteria is obtained. Step 2: Add 5-10 mL of bacterial suspension to 250 mL of liquid culture medium, place in a constant temperature shaking incubator, and repeat the operation for 4-6 cycles at a temperature of 20-30 ℃ and a rotation speed of 100-200 r / min to obtain enriched manganese oxidizing bacteria suspension. Step 3, press 101 ~10 6 The bacterial culture was diluted several times, and 100-200 µL of the diluted bacterial culture was spread on a solid culture medium containing agar using a sterilized triangular glass rod. The culture was then incubated at a constant temperature of 20-30 °C with the plate inverted until colonies of different shapes and colors appeared, thus obtaining the bacterial colonies in the water supply network.

[0043] The preparation method for solid culture medium containing agar is as follows: Add 15-20 g / L agar to the liquid culture medium, shake well, and slowly pour it into the sterilized plate to a thickness of 2-3 mm. After cooling and solidification, seal the plate and store it upside down in a constant temperature incubator.

[0044] Step 4: Use LBB colorimetric reagent to screen bacterial colonies in the water supply network. If the bacterial colonies turn blue, it indicates that they have manganese oxidizing properties, which means they are manganese oxidizing bacteria colonies.

[0045] The LBB indicator preparation method is as follows: Take 0.04~0.06g of LBB powder, dissolve it in 0.25~0.3mL of 40~50mmol / L glacial acetic acid solution, add ultrapure water, and make up to 100mL. Store at 4℃ protected from light.

[0046] The following examples further illustrate the preparation of manganese-oxidizing bacterial colonies according to the present invention.

[0047] Example 1 (1) Scrape 3 g of scale from cast iron water supply pipes and add it to 250 mL of liquid culture medium. After sealing with sealing film, place it in a constant temperature shaking incubator and carry out subculture at a temperature of 25 ℃ and a rotation speed of 150 r / min. After 5 days, manganese oxidizing bacteria suspension is obtained. (2) Add 8 mL of bacterial suspension to 250 mL of liquid culture medium, place it in a constant temperature shaking incubator, and repeat the operation for 5 cycles at a temperature of 25℃ and a rotation speed of 150 r / min to obtain enriched manganese oxidizing bacteria solution. (3) Press 10 4 The bacterial culture was diluted several times, and 150 µL of the diluted bacterial culture was spread on a solid culture medium containing agar using a sterilized triangular glass rod. The culture was then incubated at a constant temperature of 25 °C with the plate inverted until colonies of different shapes and colors appeared, thus obtaining the bacterial colonies in the water supply network.

[0048] The preparation method for solid culture medium containing agar is as follows: Add 18 g / L agar to the liquid culture medium, shake well, and slowly pour into the sterilized plate to a thickness of 2-3 mm. After cooling and solidification, seal and store upside down in a constant temperature incubator.

[0049] (4) Preparation of LBB indicator: Take 0.05g of LBB powder, dissolve it in 0.25mL of 50mmol / L glacial acetic acid solution, add ultrapure water, and make up to 100mL. Store at 4℃ protected from light.

[0050] The bacterial colonies in the water supply network were screened using LBB colorimetric reagent. If the bacterial colonies turned blue, it indicated that they had manganese oxidizing properties, and were therefore manganese oxidizing bacteria colonies.

[0051] Example 2 (1) Scrape 1g of scale from cast iron water supply pipes and add it to 200 mL of liquid culture medium. After sealing with sealing film, place it in a constant temperature shaking incubator and carry out subculture at a temperature of 30 ℃ and a rotation speed of 100 r / min. After 4 days, a suspension of manganese oxidizing bacteria is obtained. (2) Add 10 mL of bacterial suspension to 250 mL of liquid culture medium, place it in a constant temperature shaking incubator, and repeat the operation for 4 cycles at a temperature of 20℃ and a rotation speed of 100 r / min to obtain enriched manganese oxidizing bacteria solution. (3) Press 10 1 The bacterial culture was diluted several times, and 100 µL of the diluted bacterial culture was spread on a solid culture medium containing agar using a sterilized triangular glass rod. The culture was then incubated at a constant temperature of 30 °C with the plate inverted until colonies of different shapes and colors appeared, thus obtaining the bacterial colonies in the water supply network.

[0052] The preparation method for solid culture medium containing agar is as follows: Add 15 g / L agar to the liquid culture medium, shake well, and slowly pour it into the sterilized plate to a thickness of 2-3 mm. After cooling and solidification, seal the plate and store it upside down in a constant temperature incubator.

[0053] (4) Preparation of LBB indicator: Take 0.04~0.06g of LBB powder, dissolve it in 0.3mL of 45mmol / L glacial acetic acid solution, add ultrapure water, and make up to 100mL to obtain LBB indicator. Store at 4℃ protected from light.

[0054] The bacterial colonies in the water supply network were screened using LBB colorimetric reagent. If the bacterial colonies turned blue, it indicated that they had manganese oxidizing properties, and were therefore manganese oxidizing bacteria colonies.

[0055] Example 3 (1) Scrape 5 g of scale from cast iron water supply pipes and add it to 250 mL of liquid culture medium. After sealing with sealing film, place it in a constant temperature shaking incubator and carry out subculture at a temperature of 20℃ and a rotation speed of 200 r / min. After 6 days, a suspension of manganese oxidizing bacteria is obtained. (2) Add 5 mL of bacterial suspension to 250 mL of liquid culture medium, place it in a constant temperature shaking incubator, and repeat the operation for 6 cycles at a temperature of 30℃ and a rotation speed of 200 r / min to obtain enriched manganese oxidizing bacteria solution. (3) Press 10 6 The bacterial culture was diluted several times, and 200 µL of the diluted bacterial culture was spread on a solid culture medium containing agar using a sterilized triangular glass rod. The culture was then incubated at a constant temperature of 20 °C with the plate inverted until colonies of different shapes and colors appeared, thus obtaining the bacterial colonies in the water supply network.

[0056] The preparation method for solid culture medium containing agar is as follows: Add 20 g / L agar to the liquid culture medium, shake well, and slowly pour it into the sterilized plate to a thickness of 2-3 mm. After cooling and solidification, seal the plate and store it upside down in a constant temperature incubator.

[0057] (4) Preparation of LBB indicator: Take 0.04g of LBB powder, dissolve it in 0.28mL of 40mmol / L glacial acetic acid solution, add ultrapure water, and make up to 100mL to obtain LBB indicator. Store at 4℃ in the dark.

[0058] Bacterial colonies in the water supply network were screened using LBB colorimetric reagent. Colonies that turned blue indicated manganese oxidizing properties, meaning they were manganese-oxidizing bacteria. The manganese-oxidizing bacteria screened from the water supply network are shown below. Figure 1 As shown.

[0059] The following specific embodiments further illustrate the application method of the manganese-oxidizing bacterial colonies of the present invention.

[0060] Investigating manganese-oxidizing bacteria in water supply networks under different environmental factors (Mg) 2+ (For example) Growth under the influence of: Step 1: Refer to the manganese-oxidizing bacteria colonies obtained from the water supply network. On a sterile table, use a sterile inoculation loop to scrape the colonies on the manganese-oxidizing bacteria culture medium into 250 mL of sterile physiological saline and shake thoroughly to prepare a bacterial suspension. Step 2: Prepare 1500 mL of culture medium according to the manganese-oxidizing bacteria culture medium, and control the Mg content in the culture medium by adjusting the MgSO4·7H2O content. 2+ Concentration = 5 mg / L. Pour the culture medium into six 250 mL Erlenmeyer flasks. Add 10 mL of the bacterial suspension from step 1 to three of them as the experimental group, and the other three as the blank control group. Take 10 mL of the initial solution from each of the six flasks. Step 3: Measure the absorbance (OD) of the six solutions at 600 nm using a UV spectrophotometer. 600 This indicates that manganese-oxidizing bacteria thrive in Mg. 2+Initial growth at a concentration of 5 mg / L; Step 4: After sealing with sealing film, place in a constant temperature shaking incubator (temperature: 25 ℃, rotation speed: 150 r / min). Take 10 mL of sample every 12 h to measure OD. 600 value; Step 5, repeat steps 1-4 and modify Mg respectively. 2+ Concentrations of 10, 15, and 20 mg / L were calculated, and the OD values ​​were plotted with sampling time on the x-axis. 600 Plotting different Mg values ​​on the ordinate. 2+ The growth characteristics of manganese-oxidizing bacteria under the influence of concentration are shown in the figure. Figure 2 .

[0061] Depend on Figure 2 It can be seen that Mg 2+ The presence of Mg promotes the growth of manganese-oxidizing bacteria, but when Mg 2+ When the concentration reaches a certain level, the promoting effect will no longer be obvious, or may even weaken.

[0062] Investigating manganese-oxidizing bacteria in water supply networks under different environmental factors (Fe 2+ (For example) the influence of Mn 2+ Oxidation status: Step 1: Based on the manganese oxidizing bacteria colony screening, manganese oxidizing bacteria in the water supply network are obtained. On a sterile table, the colonies on the solid manganese oxidizing bacteria culture medium are scraped into 250 mL of sterile physiological saline with a sterile inoculation loop and thoroughly mixed to prepare a bacterial suspension. Step 2: Prepare 1500 mL of culture medium according to the manganese-oxidizing bacteria culture medium, and control the Fe content in the culture medium by adjusting the ferric ammonium citrate content. 2+ Concentration = 0.05 mg / L. Pour the culture medium into six 250 mL Erlenmeyer flasks. Add 10 mL of the bacterial suspension from step 1 to three of them as the experimental group, and the other three as the blank control group. Take 10 mL of the initial solution from each of the six flasks. Step 3: After filtering the six samples through a 0.22 µm microporous membrane (to prevent clogging of the flame atomic absorption spectrometer pipette), add 20 µL of nitric acid, and then determine the Mn content using a flame atomic absorption spectrometer. 2+ Concentration and manganese oxidation rate = (initial manganese content) Fe is calculated by dividing the remaining manganese content by the initial manganese content and multiplying by 100%. 2+ Initial manganese oxidation rate of manganese-oxidizing bacteria at a concentration of 0.05 mg / L; Step 4: After sealing with sealing film, place in a constant temperature shaking incubator (temperature: 25 ℃, rotation speed: 150 r / min). Take 10 mL of sample every 12 h to determine Mn. 2+The concentration was used to calculate the manganese oxidation rate; Step 5, repeat steps 1-4 and modify Fe respectively. 2+ Concentrations of 0.10, 0.20, and 0.40 mg / L were used. Finally, a plot was created with sampling time on the x-axis and manganese oxidation rate on the y-axis to show the different Fe concentrations. 2+ Manganese-oxidizing bacteria's effect on Mn concentration 2+ The oxidation rate is shown in the characteristic curve. Figure 3 .

[0063] Depend on Figure 3 It can be seen that Fe 2+ The higher the concentration, the better the effect of manganese-oxidizing bacteria on Mn. 2+ The greater the oxidation rate, the higher the Fe content. 2+ It promotes the manganese oxidation characteristics of manganese-oxidizing bacteria. The manganese oxidation rate of the manganese-oxidizing bacterial group screened in the examples can reach 99.67%.

[0064] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for preparing a culture medium for manganese-oxidizing bacteria suitable for low-carbon source environments in water supply networks, characterized in that, include: a. Prepare the culture medium according to the following mass ratio of raw materials: 2.55~2.65 parts ferric ammonium citrate, 1.95~2.05 parts MnSO4·H2O, 490~510 parts (NH4)2SO4, 190~210 parts NaCl, 250~260 parts MgSO4·7H2O, 490~510 parts K2HPO4 and 490~510 parts NaNO3; b. Adjust the pH of the culture medium to neutral, and then sterilize it under high temperature and high pressure to obtain the culture solution; c. After the culture medium has cooled, add MnSO4·H2O through a microporous filter membrane to obtain a culture medium for manganese-oxidizing bacteria.

2. The method for preparing a manganese-oxidizing bacteria culture medium suitable for low-carbon source environments in water supply networks according to claim 1, characterized in that, The pH of the culture medium was adjusted to 6.8–7.2 using HCl and NaOH solutions. Sterilize at a high temperature of 120~130℃ for 30~60 minutes.

3. The method for preparing a manganese-oxidizing bacteria culture medium suitable for low-carbon source environments in water supply networks according to claim 1, characterized in that, Cool the culture medium to 25-35 ℃, and add 4.9-5.1 mL of 300-500 mg / L MnSO4·H2O through a 0.22 µm microporous membrane on a UV-sterilized work surface.

4. A culture medium for manganese-oxidizing bacteria prepared by the method according to any one of claims 1-3, suitable for use in low-carbon source environments of water supply networks.

5. A method for screening manganese-oxidizing colonies using the manganese-oxidizing bacterial culture medium according to claim 4, characterized in that, include: a. Scrape 1-5 parts of scale from cast iron water supply pipe network, place them in 200-300 parts of liquid manganese oxidizing bacteria culture medium, and carry out subculture under constant temperature shaking to obtain manganese oxidizing bacteria suspension; b. Add 5-10 parts of bacterial suspension culture medium to 200-300 parts of liquid manganese oxidizing bacteria culture medium, and repeat the operation for several culture cycles to obtain enriched manganese oxidizing bacteria culture. c. Dilute the enriched culture of manganese-oxidizing bacteria and spread it on a solid culture medium containing agar, and incubate at a constant temperature until bacterial colonies appear; d. Use LBB indicator to screen for bacterial colonies with manganese oxidizing properties; bacterial colonies that turn blue are manganese oxidizing bacterial colonies.

6. The method for screening manganese-oxidizing colonies according to claim 5, characterized in that, Scale in cast iron water supply pipes is placed in a liquid culture medium and cultured under constant temperature shaking at 20-30 ℃ and 100-200 r / min for 4-6 days as one cycle.

7. The method for screening manganese-oxidizing colonies according to claim 5, characterized in that, The preparation method for solid culture medium containing agar is as follows: Add 15-20 g / L agar powder to the liquid culture medium, shake well, and slowly pour it into the sterilized plate to a thickness of 2-3 mm. After cooling and solidification, seal the plate and store it upside down in a constant temperature incubator.

8. The method for screening manganese-oxidizing colonies according to claim 5, characterized in that, Incubate at a constant temperature of 20-30 ℃ with the inverted incubator until colonies of different shapes and colors appear.

9. The method for screening manganese-oxidizing colonies according to claim 5, characterized in that, The bacterial culture was diluted using a serial dilution method, with a dilution factor of 10. 1 ~10 6 Diluted bacterial solutions of 100-200 µL were spread onto solid agar plates on a sterile table using sterilized pipette tips and spreaders.

10. The method for screening manganese-oxidizing colonies according to claim 5, characterized in that, The LBB colorimetric method uses LBB indicator, and the preparation method is as follows: Take 0.04~0.06 g of LBB powder by mass ratio, dissolve it in 0.25~0.30 mL of 40~50 mmol / L glacial acetic acid solution, add ultrapure water to make up to 100 mL, and store at 4 ℃ protected from light.

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