Method for sorting nitrite oxidizing bacteria based on microfluidics

Abstract

The method comprises the following steps that S1, a sample to be sorted is introduced into an inlet of a micro-fluidic chip, thalli are distributed into micro-droplets through the micro-fluidic chip, and the number of strains of each micro-droplet is smaller than or equal to 1; s2, adding a nutrient solution containing NO2 <->-N into the micro-droplets; s3, adding polyvinylpyrrolidone into the micro liquid drops, and culturing for 0.5 to 3 days; s4, monitoring the fluorescence intensity change of polyvinylpyrrolidone in the micro-droplets, and judging the generation condition of nitrate according to the fluorescence quenching degree; s5, micro-droplets generated by nitrate are collected and transferred into a shake flask to be cultured for 5-7 days; and S6, after the culture is finished, taking a culture and carrying out 16SrDNA identification. According to the method, the to-be-separated sample is subjected to micro-fluidic separation and micro-droplet culture, nitrate is qualitatively / quantitatively generated through PVP, nitrite oxidizing bacteria are obtained, and high-flux accurate separation of NOB is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism sorting, and in particular to a method for sorting nitrite oxidizing bacteria based on microfluidics. Background Art

[0002] Autotrophic nitrite oxidizing bacteria (NOB) participate in the nitrification process, converting nitrite into nitrate, and play an important role in aquaculture. Common NOB are distributed in seven genera of four bacterial phyla, including: Nitrobacter, Nirtospira, Nitrotoga, Nitrolancea, Nitromaritima, Nitrococcus and Nitrospina, but their isolation is time-consuming and labor-intensive with a low success rate.

[0003] H. LEES et al. subjected the enriched liquid to gradient centrifugation and then spread the solution to obtain Nitrobacter, but found that it was difficult to separate Nitrobacter from the associated yeast when spreading the solution on the plate. Kento Ishii et al. separated the enriched liquid using flow cytometry combined with antibiotics and only obtained one strain of Candidatus Nitrotoga. The main reasons for the difficulty in separating NOB are: (1) Slow growth: The growth generation time of autotrophic nitrite oxidizing bacteria is long, usually taking several hours or even longer to reproduce a generation, making traditional sorting very time-consuming. (2) In natural environmental samples, there are a large number of other microorganisms that may grow faster under the same culture conditions, thereby competing for limited nutrients and living space, inhibiting the growth of autotrophic NOB and increasing the difficulty of separation. Due to the lack of specific separation methods, efficient screening and separation of NOB cannot be achieved at present. Summary of the invention

[0004] In view of this, the present invention proposes a method for sorting nitrite oxidizing bacteria based on microfluidics with fast sorting speed and high sorting accuracy.

[0005] The technical solution of the present invention is implemented as follows: The present invention provides a method for sorting nitrite oxidizing bacteria based on microfluidics, comprising the following steps:

[0006] S1, introducing the sample to be sorted into the inlet of the microfluidic chip, and distributing the bacteria into each microdroplet through the microfluidic chip, and the number of strains in each microdroplet is ≤1;

[0007] S2, adding NO to the microdroplets 2 - -N nutrient solution;

[0008] S3, adding polyvinyl pyrrolidone to the microdroplets and culturing for 0.5-3 days;

[0009] S4, monitoring the fluorescence intensity change of polyvinyl pyrrolidone in the microdroplets, and judging the generation of nitrate according to the degree of fluorescence quenching;

[0010] S5, collecting the microdroplets with nitrate production and transferring them to shake flasks for culture for 5 to 7 days;

[0011] S6. After the culture is completed, the culture is taken for 16S rDNA identification.

[0012] Autotrophic NOB grow in an environment without organic matter, so the culture medium is usually inorganic. At the same time, inorganic culture medium is not conducive to the growth of heterotrophic bacteria and can play a certain screening role. 2 - -N, a part of it is oxidized to nitrate by nitrite oxidoreductase (NXR) for energy metabolism. PVP (polyvinyl pyrrolidone) has an emission peak at 360-440nm under 260-330nm wavelength excitation light. When PVP combines with nitrate under the action of electrostatic force, fluorescence quenching occurs, thereby detecting the nitrate produced by the strain, and distinguishing NOB from other non-NOB strains.

[0013] Based on the above technical solution, preferably, the nutrient solution includes NaNO 2 MgSO 4 7H 2 O.KH 2 PO 4 、FeSO 4 7H 2 O and NaCl.

[0014] Based on the above technical solution, preferably, the composition of the nutrient solution is: 100-200 mg / L NaNO 2 、40~60mg / L MgSO 4 7H 2 O, 140~160mg / L KH 2 PO 4 , 0.1~0.2mg / LFeSO 4 7H 2 O, 450-550mg / LNaCl.

[0015] On the basis of the above technical solution, preferably, the PVP solution is a PVP aqueous solution with a mass concentration of 3% to 10%.

[0016] Based on the above technical solution, preferably, in step S1, before the sample to be sorted is introduced into the microfluidic chip, the sample to be sorted is first diluted with PBS buffer to a bacterial concentration of 1×10 -3 ~5×10 -3cells / mL.

[0017] On the basis of the above technical solution, preferably, the diluted bacterial solution is placed in a centrifuge tube, ultrasonically dispersed at 35-45 kHz and 110-120 W for 2-5 min, and then introduced into the microfluidic chip.

[0018] On the basis of the above technical solution, preferably, in step S4, the change of the emission wavelength of PVP at 360-440 nm is detected under 260-330 nm excitation light.

[0019] Based on the above technical solution, preferably, in step S5, strains with a vinylpyrrolidone quenching degree greater than 50% are collected.

[0020] On the basis of the above technical solution, preferably, the sample to be sorted is a mixed bacterial liquid containing autotrophic nitrite oxidizing bacteria.

[0021] The method for sorting nitrite oxidizing bacteria based on microfluidics of the present invention has the following beneficial effects compared with the prior art:

[0022] The present invention obtains nitrite oxidizing bacteria by subjecting the sample to be separated to microfluidic separation and micro-droplet culture, and obtains nitrite oxidizing bacteria by quantitative nitrate generation through PVP, thereby achieving high-throughput and precise sorting of NOB. For Nitrobacter, the coating and picking process of the traditional separation method takes about 2 months, while the PVP-microfluidic sorting method only takes 4 to 7 days. The yield of Nitrobacter is only 4.38% when sorted without fluorescent labeling using a flow cell sorter, while PVP-microfluidic sorting can increase the yield of Nitrobacter to 60.85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The present invention is a flow chart of the microfluidic sorting of nitrite oxidizing bacteria.

[0025] Figure 2 1 is the emission spectra of PVP with different mass concentrations under 285nm excitation light. In the figure, 10% PVP, 7% PVP and 3% PVP correspond to Example 3, Example 2 and Example 1 respectively. DETAILED DESCRIPTION

[0026] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] The method for sorting nitrite oxidizing bacteria based on microfluidics in this embodiment comprises the following steps:

[0029] S1, taking a mixed bacterial solution containing autotrophic nitrite oxidizing bacteria as a sample to be separated;

[0030] S2, under sterile conditions, the mixed bacterial solution was diluted with 0.02M PBS buffer (pH = 7.2) to a bacterial concentration of 1×10 - 3 cells / mL (approximately diluted 100 times), then take 1 mL of the diluted bacterial solution into a centrifuge tube and ultrasonically disperse it at 35 kHz and 110 W for 2 min.

[0031] S3, introducing the bacterial solution after ultrasonic treatment into the inlet of the microfluidic chip, and evenly distributing the bacteria into each microdroplet through the fluid control of the microfluidic system, and the number of strains in each microdroplet is ≤1;

[0032] S4, add 10 μL of NaNO 2 Nutrient solution: 200mg / LNaNO 2 , 40mg / LMgSO 4 7H 2 O, 140mg / LKH 2 PO 4 、0.1mg / L FeSO 4 7H 2 O, 450mg / LNaCl;

[0033] S5, adding polyvinylpyrrolidone (PVP) to the microdroplets to a final concentration of 3 wt % and culturing at 28 °C for 12 h;

[0034] S6, monitoring the fluorescence intensity change of PVP in the microdroplets at an emission wavelength of 400 nm under 285 nm excitation light, and judging the generation of nitrate according to the degree of fluorescence quenching;

[0035] S7, the strains with PVP quenching degree greater than 50% were collected and transferred to shake flasks for culture for 5 days.

[0036] S8, take the culture for 16S rDNA identification.

[0037] Example 2

[0038] The method for sorting nitrite oxidizing bacteria based on microfluidics in this embodiment comprises the following steps:

[0039] S1, taking a mixed bacterial solution containing autotrophic nitrite oxidizing bacteria as a sample to be separated;

[0040] S2, under sterile conditions, the mixed bacterial solution was diluted with 0.02M PBS buffer (pH = 7.2) to a bacterial concentration of 3 × 10 - 3 cells / mL (approximately diluted 100 times), then take 1 mL of the diluted bacterial solution into a centrifuge tube and ultrasonically disperse it at 40 kHz and 115 W for 3.5 min;

[0041] S3, introducing the bacterial solution after ultrasonic treatment into the inlet of the microfluidic chip, and evenly distributing the bacteria into each microdroplet through the fluid control of the microfluidic system, and the number of strains in each microdroplet is ≤1;

[0042] S4, add 10 μL of NaNO 2 Nutrient solution: 150mg / LNaNO 2 、40~60mg / L MgSO 4 7H 2 O, 150mg / LKH 2 PO 4 、0.15mg / LFeSO 4 7H 2 O, 500 mg / L NaCl;

[0043] S5, adding polyvinylpyrrolidone (PVP) to the microdroplets to a final concentration of 7 wt % and culturing at 28 °C for 40 h;

[0044] S6, monitoring the fluorescence intensity change of PVP in the microdroplets at an emission wavelength of 400 nm under 285 nm excitation light, and judging the generation of nitrate according to the degree of fluorescence quenching;

[0045] S6, strains with a PVP quenching degree greater than 50% were collected and transferred to shake flasks for 6 days;

[0046] S7, take the culture for 16S rDNA identification.

[0047] Example 3

[0048] The method for sorting nitrite oxidizing bacteria based on microfluidics in this embodiment comprises the following steps:

[0049] S1, taking a mixed bacterial solution containing autotrophic nitrite oxidizing bacteria as a sample to be separated;

[0050] S2, under sterile conditions, the mixed bacterial solution was diluted with 0.02M PBS buffer (pH = 7.2) to a bacterial concentration of 5×10 - 3 cells / mL (approximately diluted 100 times), then take 1 mL of the diluted bacterial solution into a centrifuge tube and ultrasonically disperse it at 45 kHz and 120 W for 5 min;

[0051] S3, introducing the bacterial solution after ultrasonic treatment into the inlet of the microfluidic chip, and evenly distributing the bacteria into each microdroplet through the fluid control of the microfluidic system, and the number of strains in each microdroplet is ≤1;

[0052] S4, add 10 μL of NaNO 2 Nutrient solution: 100mg / LNaNO 2 、60mg / LMgSO 4 7H 2 O, 160mg / LKH 2 PO 4 、0.2mg / L FeSO 4 7H 2 O, 550mg / LNaCl;

[0053] S5, adding polyvinylpyrrolidone (PVP) to the microdroplets to a final concentration of 10 wt % and culturing at 28 °C for 72 h;

[0054] S6, monitoring the fluorescence intensity change of PVP in the microdroplets at an emission wavelength of 400 nm under 285 nm excitation light, and judging the generation of nitrate according to the degree of fluorescence quenching;

[0055] S7, strains with PVP quenching greater than 50% were collected and transferred to shake flasks for 7 days;

[0056] S8, take the culture for 16S rDNA identification.

[0057] Comparative Example 1

[0058] Comparative Example 1 Compared with Example 1, no PVP was added, and the specific steps were as follows:

[0059] The method for separating nitrite oxidizing bacteria based on microfluidics in this comparative example comprises the following steps:

[0060] S1, taking a mixed bacterial solution containing autotrophic nitrite oxidizing bacteria as a sample to be separated;

[0061] S2, under sterile conditions, the mixed bacterial solution was diluted with 0.02M PBS buffer (pH = 7.2) to a bacterial concentration of 1×10 - 3 cells / mL (approximately diluted 100 times), then take 1 mL of the diluted bacterial solution into a centrifuge tube and disperse it ultrasonically at 35 kHz and 110 W for 2 min;

[0062] S3, introducing the bacterial solution after ultrasonic treatment into the inlet of the microfluidic chip, and evenly distributing the bacteria into each microdroplet through the fluid control of the microfluidic system, and the number of strains in each microdroplet is ≤1;

[0063] S4, add 10 μL of NaNO 2 Nutrient solution: 200mg / LNaNO 2 , 40mg / LMgSO 4 7H 2 O, 140mg / LKH 2 PO 4 、0.1mg / LFeSO 4 7H 2 O, 450 mg / L NaCl, culture at 28°C for 12 h;

[0064] S5, transferring the above culture into a shake flask for 5 days;

[0065] S6. Use the national standard method (HJ / T346-2007) to detect nitrate production in all cultures, and perform 16S rDNA identification on cultures that produce nitrate.

[0066] Comparative Example 2

[0067] Compared with Example 1, Comparative Example 2 has a different culture medium, and the specific steps are as follows:

[0068] The method for separating nitrite oxidizing bacteria based on microfluidics in this comparative example comprises the following steps:

[0069] S1, taking a mixed bacterial solution containing autotrophic nitrite oxidizing bacteria as a sample to be separated;

[0070] S2, under sterile conditions, the mixed bacterial solution was diluted with 0.02M PBS buffer (pH = 7.2) to a bacterial concentration of 1×10 - 3 cells / mL (approximately diluted 100 times), then take 1 mL of the diluted bacterial solution into a centrifuge tube and disperse it ultrasonically at 35 kHz and 110 W for 2 min;

[0071] S3, introducing the bacterial solution after ultrasonic treatment into the inlet of the microfluidic chip, and evenly distributing the bacteria into each microdroplet through the fluid control of the microfluidic system, and the number of strains in each microdroplet is ≤1;

[0072] S4, add 10 μL of NaNO 2 Nutrient solution: 200mg / LNaNO 2 , 10g / L tryptone, 5g / L yeast extract, 450mg / L NaCl;

[0073] S5, adding polyvinylpyrrolidone (PVP) to the microdroplets to a final concentration of 3 wt % and culturing at 28 °C for 12 h;

[0074] S6, monitoring the fluorescence intensity change of PVP in the microdroplets at an emission wavelength of 400 nm under 285 nm excitation light, and judging the generation of nitrate according to the degree of fluorescence quenching;

[0075] S7, strains with a PVP quenching degree greater than 50% were collected and transferred to shake flasks for 5 days;

[0076] S8, take the culture for 16S rDNA identification.

[0077] Comparative Example 3

[0078] Compared with Example 1, Comparative Example 3 has a different PVP concentration, specifically 1 wt %, and the rest of the contents are the same as Example 1.

[0079] Comparative Example 4

[0080] Compared with Example 1, Comparative Example 4 has a different PVP concentration, specifically 15 wt %, and the rest of the contents are the same as Example 1.

[0081] Table 1 Sorting comparison

[0082] time Nitrobacter yield Example 1 5～6d 60.85％ Example 2 7～8d 58.74％ Example 3 10d 59.92％ Comparative Example 1 60d 50.09％ Comparative Example 2 10d 0％ Comparative Example 3 5～6d 4.34％ Comparative Example 4 18d 44.75％

[0083] As shown in Table 1, the method of sorting nitrite oxidizing bacteria based on microfluidics of the present invention has the advantages of short time and high yield of Nitrobacter. Among them, Example 1 has the best effect, and the yield of Nitrobacter reaches 60.85%. Under the same sorting time, the yield of Nitrobacter by other sorting methods is significantly lower than that of the embodiment of the present invention. Changing the culture medium or changing the amount of PVP will reduce the yield of Nitrobacter.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for sorting nitrite oxidizing bacteria based on microfluidics, characterized in that: The steps include: S1, introducing the sample to be sorted into the inlet of the microfluidic chip, and distributing the bacteria into each microdroplet through the microfluidic chip, and the number of strains in each microdroplet is ≤1; S2, adding NO2 - -N nutrient solution; S3, adding polyvinyl pyrrolidone to the microdroplets and culturing for 0.5-3 days; S4, monitoring the fluorescence intensity change of polyvinyl pyrrolidone in the microdroplets, and judging the generation of nitrate according to the degree of fluorescence quenching; S5, collecting the microdroplets with nitrate production and transferring them to shake flasks for culture for 5 to 7 days; S6. After the culture is completed, the culture is taken for 16S rDNA identification.

2. A method for sorting nitrite oxidizing bacteria based on microfluidics according to claim 1, characterized in that: The nutrient solution includes NaNO2, MgSO4·7H2O, KH2PO4, FeSO4·7H2O and NaCl.

3. A method for sorting nitrite oxidizing bacteria based on microfluidics as claimed in claim 2, characterized in that: The components of the nutrient solution are: 100-200 mg / L NaNO2, 40-60 mg / LMgSO4·7H2O, 140-160 mg / L KH2PO4, 0.1-0.2 mg / LFeSO4·7H2O, 450-550 mg / L NaCl.

4. A method for sorting nitrite oxidizing bacteria based on microfluidics according to claim 1, characterized in that: The PVP solution is a PVP aqueous solution with a mass concentration of 3% to 10%.

5. A method for sorting nitrite oxidizing bacteria based on microfluidics according to claim 1, characterized in that: In step S1, before the sample to be sorted is introduced into the microfluidic chip, the sample to be sorted is diluted with PBS buffer to a bacterial concentration of 1×10 -3 ~5×10 -3 cells / mL.

6. A method for sorting nitrite oxidizing bacteria based on microfluidics according to claim 5, characterized in that: The diluted bacterial solution was placed in a centrifuge tube, and ultrasonically dispersed for 2 to 5 minutes at 35 to 45 kHz and 110 to 120 W, and then introduced into the microfluidic chip.

7. A method for sorting nitrite oxidizing bacteria based on microfluidics as claimed in claim 1, characterized in that: In step S4, the change of the emission wavelength of PVP at 400 nm is detected under 285 nm excitation light.

8. A method for sorting nitrite oxidizing bacteria based on microfluidics as claimed in claim 1, characterized in that: In step S5, strains with a PVP quenching degree greater than 50% are collected.

9. A method for sorting nitrite oxidizing bacteria based on microfluidics according to claim 1, characterized in that: The sample to be sorted is a mixed bacterial liquid containing autotrophic nitrite oxidizing bacteria.

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