Sulfur-oxidizing bacteria high-throughput screening method and application

Abstract

The invention belongs to the technical field of strain screening, discloses a high-throughput screening method for sulfur-oxidizing bacteria and application, and particularly discloses application of 7-amino-4-methylcoumarin to screening of the sulfur-oxidizing bacteria or preparation of products for screening of the sulfur-oxidizing bacteria. The invention discloses the application of 7-amino-4-methylcoumarin in screening sulfur-oxidizing bacteria for the first time, and rapid, convenient, high-throughput and high-efficiency screening of sulfur-oxidizing bacteria can be realized through 7-amino-4-methylcoumarin.

Description

Technical Field

[0001] The invention belongs to the technical field of strain screening, and specifically relates to a high-throughput screening method for sulfur oxidizing bacteria and an application thereof. Background Art

[0002] Sulfur, an essential element for life, is ubiquitous in nature. The number of outer electrons of sulfur atoms reaches six, giving it a rich valence state, including elemental sulfur (0 valence), reduced sulfur (-2 valence, +2 valence, +4 valence) and sulfate (+6 valence). Common reduced sulfur forms include sulfide, thiosulfate and sulfite.

[0003] Hydrogen sulfide, a gas known for its pungent smell of rotten eggs, is produced in a variety of ways, including industrial activities such as leather making, brewing and rubber manufacturing, as well as animal husbandry in agriculture and even sewage discharged in daily life. It is a hotbed for hydrogen sulfide production and is widely distributed, posing a serious threat to the environment, humans and animals, especially aquatic life. It easily reacts with metal ions such as copper (II) ions, iron (II) ions and manganese (II) ions in water to form metal sulfides. This process not only darkens the color of the water body, but also produces unpleasant odors, seriously damaging the urban environment and having a negative impact on people's physical and mental health. Low concentrations of hydrogen sulfide can irritate the eyes, nervous system and respiratory system, causing symptoms such as headaches, dizziness and fatigue. High concentrations of hydrogen sulfide may cause shock or even death. In aquaculture, the toxic effect of hydrogen sulfide is particularly significant. According to my country's "Fishery Water Quality Standards", the concentration of sulfide in water bodies should be strictly controlled below 0.2mg / L. Once the concentration of hydrogen sulfide in the water exceeds the standard, it will not only cause breathing difficulties for aquatic animals such as fish, shrimp and crabs, but will also slow down their growth rate, reduce their disease resistance, and may even cause acute death.

[0004] Therefore, oxidizing sulfide ions into more stable sulfate ions is a key strategy to reduce their toxicity. However, relying on indigenous microorganisms in the natural environment to oxidize sulfide is usually a very slow process, often slower than the input rate of exogenous sulfide, resulting in the accumulation of sulfide. In contrast, the microbial method provides a solution that is both non-secondary pollution, targeted and cost-effective. Sulfur-oxidizing bacteria (SOB) are a class of microorganisms that can oxidize sulfide during growth and metabolism to obtain energy. Therefore, screening and isolating efficient sulfur-oxidizing bacteria plays a vital role in eliminating the hazards of sulfide.

[0005] The currently commonly used sulfur-oxidizing strain screening and separation technology usually follows the following steps: ① Collect environmental samples; ② Use enrichment culture medium to enrich the samples and spread them on plate culture medium; ③ Select the growing colonies from the plate for streaking separation and purification; ④ Transfer the purified single colony to a larger volume of culture medium containing sulfide. After a period of cultivation, use the industry standard methylene blue spectrophotometry or iodine titration to measure the change in sulfide concentration to verify whether the selected strain has sulfur oxidation ability. It can be seen that only on the basis of a sufficiently large screening base can the probability of screening sulfur-oxidizing bacteria with excellent properties be increased. Therefore, high-throughput screening and identification of sulfur oxidation function methods are the key to obtaining strains with excellent properties. However, the current screening and separation methods are complicated to operate and cannot complete screening and separation with high efficiency and high throughput. Therefore, this application is specially proposed. Summary of the invention

[0006] The present invention aims to propose an innovative method for screening sulfur-oxidizing strains, which is not only simple and quick to operate, but also has the characteristics of high efficiency and high throughput, and can significantly improve efficiency and accuracy. The introduction of this method will improve the convenience and efficiency of screening sulfur-oxidizing strains.

[0007] The purpose of the first aspect of the present invention is to provide the use of 7-amino-4-methylcoumarin in screening sulfur oxidizing bacteria or preparing products for screening sulfur oxidizing bacteria.

[0008] The second aspect of the present invention aims to provide a method for screening sulfur oxidizing bacteria.

[0009] The third aspect of the present invention aims to provide an application of the screening method of the second aspect of the present invention in evaluating the sulfur oxidation function of sulfur oxidizing bacteria.

[0010] The fourth aspect of the present invention aims to provide a method for evaluating the sulfur oxidation function of a strain.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides the use of 7-amino-4-methylcoumarin in screening sulfur oxidizing bacteria or preparing a product for screening sulfur oxidizing bacteria.

[0013] A second aspect of the present invention provides a method for screening sulfur oxidizing bacteria, comprising the following steps:

[0014] (1) The strain to be tested is cultured in a medium containing sulfide, and physiological saline is used to replace the strain to be tested as a blank control group;

[0015] (2) adding 7-amino-4-methylcoumarin to the culture system of step (1) and culturing;

[0016] (3) Determine the fluorescence intensity of the culture system after culturing in step (2), and screen out sulfur oxidizing bacteria by comparing it with the fluorescence intensity of the blank control group.

[0017] In some embodiments of the present invention, the final concentration of 7-amino-4-methylcoumarin in the culture system is 5 to 30 μM.

[0018] In some embodiments of the present invention, the final concentration of 7-amino-4-methylcoumarin in the culture system is 10 to 30 μM.

[0019] In some embodiments of the present invention, the final concentration of 7-amino-4-methylcoumarin in the culture system is 10-20 μM.

[0020] In some embodiments of the present invention, the final concentration of sulfide in the sulfide-containing culture medium is 100-300 μM.

[0021] In some embodiments of the present invention, the final concentration of sulfide in the sulfide-containing culture medium is 100-250 μM.

[0022] In some embodiments of the present invention, the final concentration of sulfide in the sulfide-containing culture medium is 100-200 μM.

[0023] In some embodiments of the present invention, the sulfide comprises sodium sulfide or sodium hydrosulfide.

[0024] In some embodiments of the present invention, the culture conditions of step (1) are static culture at 27-35° C. for 1-3 hours.

[0025] In some embodiments of the present invention, the culture conditions of step (1) are static culture at 30-35° C. for 1-2 hours.

[0026] In some embodiments of the present invention, the culture conditions of step (1) are static culture at 30-35° C. for 1-1.5 h.

[0027] In some embodiments of the present invention, the culture conditions of step (2) are static culture at 30-40° C. for 1-2 hours.

[0028] In some embodiments of the present invention, the culture conditions of step (2) are static culture at 30-37° C. for 1-2 hours.

[0029] In some embodiments of the present invention, the culture conditions of step (2) are static culture at 30-37° C. for 1-1.5 h.

[0030] In some embodiments of the present invention, the strains to be tested include original screening bacteria isolated from nature and improved strains obtained through artificial mutagenesis or genetic modification techniques.

[0031] In some embodiments of the present invention, the fluorescence intensity is measured using an enzyme reader.

[0032] In some embodiments of the present invention, when the microplate reader is used for determination, the excitation wavelength is 365 nm and the emission wavelength is 450 nm.

[0033] In some embodiments of the present invention, the container for culturing the strain comprises a black transparent bottom culture plate.

[0034] In some embodiments of the present invention, when the fluorescence intensity of the test strain is lower than that of the blank control group, it indicates that the test strain has sulfur oxidation function, and the smaller the fluorescence intensity, the stronger the sulfur oxidation function of the test strain.

[0035] The strains to be screened involved in the present invention include both the original screening products isolated from nature and the improved strains obtained by artificial mutagenesis or genetic modification technology. During the screening process, parameters such as culture time, culture conditions, and sulfide-containing culture medium can be selected or adjusted according to the research objectives and needs. The adoption, adjustment or optimization of these parameters are based on the guidance of the prior art and belong to the professional skills of those skilled in the art. The method of the present invention is applicable to all strains with sulfur oxidation function, regardless of their source, and efficient screening can be achieved through this process.

[0036] The third aspect of the present invention provides the use of the screening method of the second aspect of the present invention in evaluating the sulfur oxidation function of sulfur oxidizing bacteria.

[0037] A fourth aspect of the present invention provides a method for evaluating the sulfur oxidation function of a strain, comprising the steps of adopting the method of the second aspect of the present invention.

[0038] The beneficial effects of the present invention are:

[0039] The present invention discloses for the first time the application of 7-amino-4-methylcoumarin in screening sulfur-oxidizing bacteria, and can realize rapid, convenient, high-throughput and high-efficiency screening of sulfur-oxidizing bacteria through 7-amino-4-methylcoumarin.

[0040] Compared with traditional screening methods (large workload and low efficiency), the present invention aims to break through the limitations of traditional screening methods and provide a screening scheme for sulfur oxidizing bacteria. This method can not only improve the screening flux and efficiency, but also has the characteristics of simple and quick operation and low workload. It improves the convenience and efficiency of the screening of sulfur oxidizing strains as a whole, provides strong technical support for the research and application of sulfur oxidizing bacteria, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the standard curve of fluorescence intensity and sulfide concentration. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below through specific examples.

[0043] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0046] Example 1

[0047] 7-Amino-4-methylcoumarin (CAS: 26093-31-2) can be used as a fluorescent probe sensitive to hydrogen sulfide for verification. The specific process is as follows:

[0048] Prepare the sodium hydrosulfide aqueous solution and filter and sterilize. Add 140 μL of inorganic salt culture medium to a 96-well black plate, and then add 20 μL of 7-amino-4-methylcoumarin to make the final concentration of the probe 10 μM. Then add 20 μL of sodium hydrosulfide aqueous solution respectively (the sulfide concentration must be calibrated by the method in Appendix A of HJ 1226-2021) to make the sulfide concentrations of 0, 10, 25, 50, 75, and 100 μM, respectively. Set up 2 parallel test wells for each sulfide concentration. Cover the multiwell plate cover. Place the multiwell plate in a 37°C constant temperature incubator and incubate for 1 hour. After the incubation is completed, use a multifunctional microplate reader to detect the fluorescence intensity under excitation light of 365 nm and emission light of 450 nm (top detection, gain 100, light source xenon flash lamp).

[0049] As shown in Table 1 and Figure 1 As shown, the correlation coefficient between sulfide concentration and fluorescence intensity is 0.9987, which has a good correlation, indicating that 7-amino-4-methylcoumarin can be used to quickly detect the concentration of sulfide.

[0050] Table 1 Relationship between sulfide concentration and fluorescence intensity

[0051] Sulfide concentration (μM) Fluorescence intensity 1 Fluorescence intensity 2 Mean fluorescence intensity 0 2458 2654 2556 10 7545 8425 7985 25 19595 20627 20111 50 37401 35311 36356 75 49218 54349 51783.5 100 70345 71536 70940.5

[0052] Example 2

[0053] A method for screening sulfur oxidizing bacteria comprises the following steps:

[0054] (1) Obtain a single strain obtained through screening or mutagenesis.

[0055] (2) Pick a single strain and culture it in a deep-well plate containing 1 mL of LB liquid medium at 28-30°C, 180-200 rpm, and for 16-20 hours. Remove the medium, resuspend with saline, centrifuge, wash three times, and resuspend with saline to obtain a bacterial solution.

[0056] (3) The bacterial solution was added to a black 96-well plate and cultured in a medium containing 100-200 μM sulfide (2 g / L potassium dihydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 0.4 g / L ammonium chloride, 0.01 g / L ferrous sulfate heptahydrate, and after sterilization, a filtered and sterilized Na2S solution was added). A blank control group was set up at the same time, in which the bacterial solution was replaced with the same volume of physiological saline. Two test wells were used as parallel for each treatment.

[0057] (4) Add 10 μM to 20 μM 7-amino-4-methylcoumarin to the above mixed culture medium for culturing at 30° C. to 37° C. for 1 to 1.5 h.

[0058] (5) Use an ELISA reader with full-wavelength fluorescence detection function to measure the fluorescence intensity of each test well at an excitation wavelength of 365 nm and an emission wavelength of 450 nm. The fluorescence intensity value measured in the test well is lower than the fluorescence intensity value of the blank control well, indicating that the bacteria in the well have sulfur oxidation function. The lower the fluorescence intensity value, the stronger the sulfur oxidation ability of the bacteria in the test well.

[0059] Example 3

[0060] The method of Example 2 was used to screen sulfur oxidizing bacteria, and the specific process was as follows:

[0061] (1) Obtain a single strain obtained through screening or mutagenesis: Collect the black and smelly sludge from the fish pond. Weigh 10g of sludge, mix it with 90mL of sterile saline, and add sterile glass beads to enhance the mixing effect. Ensure that the sludge and saline are fully mixed by continuous oscillation for 30 minutes to release the microorganisms therein. After mixing evenly, inoculate the suspension into the enrichment medium and place it in a constant temperature incubator at 30°C for 3 days. As the culture proceeds, when it is observed that the culture medium becomes obviously turbid, indicating that the microorganisms have begun to multiply in large numbers, the next step of strain isolation can be carried out.

[0062] The enriched bacterial solution was concentrated at 10 -1 , 10 -2, 10 -3 After gradually diluting the concentration by 100 times, evenly spread it on the plate. Place the plate in a 30℃ constant temperature incubator and culture for 1-2 days. Pick colonies with different morphologies and purify them by streaking 2-3 times to obtain purified strains.

[0063] (2) The purified strain obtained above was inoculated into a 48-well deep-well plate, and 1 mL of LB medium was added to each well. The culture was incubated at 180 rpm and 30°C for 16 to 20 h. Then 500 μL of bacterial solution was taken into a 2 mL Eppendorf tube, the medium was removed, the tube was resuspended with physiological saline, centrifuged, and washed three times, and resuspended with physiological saline to obtain a bacterial solution.

[0064] (3) Add the bacterial solution resuspended in the above-mentioned physiological saline to a culture medium containing a sulfide concentration of 100 μM, and the total volume of the bacterial solution and the culture medium is 180 μL. Place it in a 30°C constant temperature incubator and incubate it for 1 hour, then add 20 μL of 7-amino-4-methylcoumarin to make the final concentration of the probe 10 μM. Set up 2 parallels for each bacterium, and set up a blank control at the same time (using physiological saline instead of bacterial solution). Place it at 37°C and incubate it for 1 hour. After the culture is completed, use a multifunctional microplate reader (with full-wavelength fluorescence detection function) to detect the fluorescence intensity under the conditions of excitation light 340nm and emission light 450nm (top detection, gain 100, light source xenon flash lamp).

[0065] Table 2 Fluorescence intensity of mixed cultures in multiwell plates

[0066]

[0067]

[0068] (4) Select the bacteria with lower average fluorescence intensity than the blank control group in Table 2, inoculate them into a deep well plate containing 1 mL of LB medium, and culture them at 30° C. and 180 rpm for 16 to 18 h to screen out sulfur oxidizing bacteria with better sulfur oxidation effect.

[0069] The above sulfur oxidizing bacteria were verified using a traditional method, as follows: the cultured strain was resuspended in sterile physiological saline, inoculated into 950 μL sulfide medium (sulfide final concentration was 100 mg / L) at a 5% inoculum (V / V), cultured at 30°C and 120 rpm for 48 hours, and the sulfide concentration after 48 hours was detected by methylene blue spectrophotometry to calculate the sulfur oxidation rate of the bacteria. The calculation formula is as follows:

[0070]

[0071] Table 3 Sulfur oxidation rate of selected strains

[0072] Strain number Sulfur oxidation rate Q31 100.00％ Q14 100.00％ Q19 81.26％ BO-4 35.00％ BO-3 47.00％ H2O-4-3 45.00％

[0073] Verified by traditional methods, it can be seen from Table 3 that the 6 strains with fluorescence intensity lower than the blank control all have sulfur oxidation function; and the smaller the fluorescence intensity of the bacteria, the higher the verified sulfur oxidation rate. Among them, Q31 and Q14 could not detect sulfide after reacting with sulfide for 48 hours, and it is believed that their oxidation rate reached 100% after 48 hours.

[0074] In summary, the method of the present invention has been verified, showing its stability and reliability, and the operation process is concise and clear. In contrast, the traditional method uses strains to oxidize sulfides in shake flasks, and then determines the sulfide concentration according to the HJ 1226-2021 standard to evaluate the sulfur oxidation ability and efficiency of the strains. However, this method not only has a complicated operation process and a large amount of reagents, but also requires frequent updates of the standard curve. In contrast, the method of the present invention allows direct operation in a 96-well plate, greatly reducing the amount of reagents used, and can screen multiple strains of bacteria at one time. Therefore, the method of the present invention has the significant advantage of high-throughput screening, and can efficiently identify sulfur-oxidizing bacteria with excellent performance.

[0075] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of 7-amino-4-methylcoumarin in screening sulfur-oxidizing bacteria or preparing products for screening sulfur-oxidizing bacteria.

2. A method for screening sulfur oxidizing bacteria, comprising the following steps: (1) The strain to be tested is cultured in a medium containing sulfide, and physiological saline is used to replace the strain to be tested as a blank control group; (2) adding 7-amino-4-methylcoumarin to the culture system of step (1) and culturing; (3) Determine the fluorescence intensity of the culture system after culturing in step (2), and screen out sulfur oxidizing bacteria by comparing it with the fluorescence intensity of the blank control group.

3. The screening method according to claim 2, characterized in that The final concentration of the 7-amino-4-methylcoumarin in the culture system is 5 to 30 μM.

4. The screening method according to claim 3, characterized in that The final concentration of sulfide in the sulfide-containing culture medium is 100-300 μM.

5. The screening method according to any one of claims 2 to 4, characterized in that The culture conditions of step (1) are static culture at 27-35° C. for 1-3 hours.

6. The screening method according to any one of claims 2 to 4, characterized in that The culture conditions of step (2) are static culture at 30-40° C. for 1-2 hours.

7. The screening method according to any one of claims 2 to 4, characterized in that The strains to be tested include original screening bacteria separated from nature and improved strains obtained through artificial mutagenesis or gene modification technology.

8. The screening method according to any one of claims 2 to 4, characterized in that The fluorescence intensity was measured using an enzyme-labeled instrument.

9. The screening method according to claim 8, characterized in that When the fluorescence intensity of the strain to be tested is lower than that of the blank control group, it indicates that the strain to be tested has sulfur oxidation function, and the smaller the fluorescence intensity, the stronger the sulfur oxidation function of the strain to be tested.

10. Use of the screening method according to any one of claims 2 to 9 in evaluating the sulfur oxidation function of a strain.

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