Method for verifying performance of microorganism culture in microfluidic droplets

Abstract

The application provides a method for obtaining the culture performance of target microorganisms in a microdroplet system, which comprises the following steps: activating and diluting target microorganisms to obtain target bacterial liquid; culturing the target bacterial liquid by using a microdroplet culture system; and obtaining the culture performance of the target microorganisms in the microdroplet system according to the culture performance relationship of the target microorganisms in the microdroplet system and a traditional culture system. According to the growth data of the microorganisms in the existing traditional culture system, the accuracy of the optical method (OD measurement) commonly used in the microdroplet culture system can be calibrated and verified, and the culture condition of the microorganisms in the microdroplet system can be more accurately predicted.

Description

Technical Field

[0001] This application belongs to the field of microbial engineering technology, specifically relating to a method for verifying the performance of microbial culture within microfluidic droplets. Background Technology

[0002] Droplet microfluidics is a major branch of microfluidics, referring to the technology of precisely controlling extremely small volumes of liquid flow. Microbial culture technology based on droplet microfluidics can avoid the influence of interspecies competition by placing a small group or even a single microorganism in a droplet for cultivation, thereby achieving the goal of precisely controlling the growth environment of microorganisms and isolating microorganisms that cannot be cultured by traditional methods.

[0003] In existing technologies, the conventional method for obtaining microbial growth curves in microfluidic droplet culture systems is optical measurement, particularly by measuring the optical density (OD) of the droplets to indirectly reflect the biomass of the microorganisms. This method typically integrates an optical detection module into the microfluidic system. When a droplet containing microorganisms flows through this module, light of a specific wavelength (typically 600 nm, i.e., OD) is detected. 600 By passing through the droplet and detecting changes in the intensity of transmitted or scattered light, the OD value can be calculated. By continuously or periodically monitoring the changes in the OD value of the droplet, a curve reflecting the growth trend of the microbial community can be plotted.

[0004] However, existing technologies employ optical measurement methods, such as OD... 600 The following two core problems exist in monitoring microbial growth curves: (1) Inability to accurately monitor the early growth stage: Due to the detection limit of optical measurement methods, it is impossible to effectively capture the growth dynamics of microorganisms in the lag phase and the early stage of exponential growth, resulting in the loss of key information in the growth curve; (2) The monitoring results are indirect and easily interfered with relative values: As an indirect indicator, the correlation between OD value and the actual number of microorganisms (cfu) is affected by various factors such as strain characteristics, culture medium composition and growth stage, resulting in reduced accuracy and comparability of monitoring results. Therefore, this method cannot be directly compared with traditional culture methods. Summary of the Invention

[0005] To address the problems mentioned above regarding the use of optical measurement methods to monitor microbial growth curves when culturing microorganisms in droplet microfluidic systems, this application provides a method for obtaining the culture performance of target microorganisms in microdroplet systems.

[0006] Specifically, the invention involves the following: 1. A method for obtaining the culture performance of a target microorganism in a microdroplet culture system, comprising: The target microorganism is activated and diluted to obtain the target bacterial solution; The target bacterial culture was cultured using a microdroplet culture system; Based on the relationship between the culture performance of the target microorganism in the microdroplet culture system and the traditional culture system, the culture performance of the target microorganism in the microdroplet culture system was obtained.

[0007] 2. The method according to item 1, wherein the culture performance includes the maximum bacterial concentration lg value (A) and the time to reach A (T). 95 ), maximum specific growth rate (μ max ), delay time (λ).

[0008] 3. The method according to item 1, wherein the relationship between the culture performance of the target microorganism in the microdroplet culture system and the conventional culture system is obtained through the following steps: The target microorganism is activated and diluted to obtain the target bacterial solution; The target bacterial culture was cultured using both a microdroplet culture system and a traditional culture system. At a given time, the concentration (cfu / mL) of the target microorganism in the microdroplet culture system and the conventional culture system was obtained. Based on the lg value of the concentration at the given time, the relationship between the culture performance of the target microorganism in the microdroplet culture system and the traditional culture system was obtained using the Gompertz model.

[0009] 4. The method according to item 3, wherein the given time includes: 0h, 2h, 3h, 4h, 6h, 8h, 23h, 24h.

[0010] 5. The method according to item 3, wherein obtaining the concentration of the target microorganism in the microdroplet culture system includes the following steps: Collect microdroplets at the given time. The microdroplet structure was disrupted to obtain a sample containing the target microorganism; The above samples were transferred to a culture medium for incubation to obtain the concentration of the target microorganism in the microdroplet culture system at the given time.

[0011] 6. The method according to item 5, wherein the process of collecting microdroplets includes: introducing microdroplets into a culture tube, cutting off a tube segment containing microdroplets, and collecting the microdroplets within the tube segment. The pipe section is 1-3cm, preferably 2cm.

[0012] 7. The method according to item 5, wherein obtaining the sample containing the target microorganism comprises: using a demulsifier to disrupt the microdroplet structure, followed by filtration, to obtain the sample containing the target microorganism. The demulsifier is selected from one or more of Tween 80, Tween 65, Span 80, Span 60 or Span 20, or one or more of Tween 80, Tween 65, Span 80, Span 60 or Span 20 combined with lecithin.

[0013] 8. The method according to item 3, wherein the Gompertz model uses the following formula to obtain the relationship between the culture performance of the target microorganism in the microdroplet culture system and the conventional culture system: (Formula 1) (Formula 2) In formula 1 or formula 2, Y(t) represents the logarithmic value of the target microbial concentration at time t in a microdroplet culture system or a traditional culture system, with base 10 [lg(cfu / mL)]. Y0 represents the logarithmic value of the initial concentration of the target microorganism in a microdroplet culture system or a traditional culture system, with the base 10 [lg(cfu / mL)]. A represents the logarithmic value of the maximum concentration of the target microorganism in a microdroplet culture system or a traditional culture system, with the base 10 [lg(cfu / mL)]. μ max This indicates the maximum specific growth rate (h) of the target microorganism in a microdroplet culture system or a conventional culture system. -1 ); λ represents the lag time (h) of the target microorganism in the microdroplet culture system or the traditional culture system. T 95 This indicates the time (h) it takes for the target microorganism to reach A in a microdroplet culture system or a traditional culture system.

[0014] 9. The method according to any one of items 1-8, wherein, The target microorganism is Escherichia coli. The A value of *Escherichia coli* in the microdroplet culture system differed significantly from that in the conventional culture system; μ in the microdroplet culture system max Compared with μ in traditional culture systems max No significant difference; λ in the microdroplet culture system was not significantly different from that in the conventional culture system; T in the microdroplet culture system 95 Compared with the T in the traditional cultivation system 95 There are significant differences; Alternatively, the target microorganism may be Bacillus subtilis. The A value of Bacillus subtilis in the microdroplet culture system differed significantly from that in the conventional culture system; μ in the microdroplet culture system maxCompared with μ in traditional culture systems max No significant difference; λ in the microdroplet culture system was significantly different from that in the traditional culture system; T in the microdroplet culture system 95 Compared with the T in the traditional cultivation system 95 There are significant differences.

[0015] 10. The method according to item 9, wherein, The A value of Escherichia coli in the microdroplet culture system is 0.5-1 lg (cfu / mL) greater than that in the conventional culture system. The Escherichia coli in the microdroplet culture system T 95 For: T under the traditional training system 95 Arrive 6-8 hours in advance; The A value of Bacillus subtilis in the microdroplet culture system is 1-3 lg (cfu / mL) greater than that in the conventional culture system. The λ of Bacillus subtilis in the microdroplet culture system is 0.5-1 h shorter than that in the conventional culture system; The Bacillus subtilis in the microdroplet culture system T 95 For: T under the traditional training system 95 Please arrive 4-6 hours in advance.

[0016] 11. The method according to item 10, wherein, The A value in the Escherichia coli microdroplet culture system was 9.779 ± 0.05 lg (cfu / mL), while the A value in the conventional culture system was 9.056 ± 0.16 lg (cfu / mL); the μ value in the microdroplet culture system was... max It is 0.8917±0.03h -1 μ under traditional culture system max It is 0.8616±0.08h -1 The λ value in the microdroplet culture system was 1.288±0.09 h, while that in the conventional culture system was 0.7663±0.42 h; the T value in the microdroplet culture system was... 95 The time to T was 11.18 ± 0.20 h under the traditional culture system. 95 It was 17.96 ± 1.80 h; The A value of *Bacillus subtilis* in the microdroplet culture system was 9.131 ± 0.11 lg (cfu / mL), while the A value in the conventional culture system was 7.12 ± 0.03 lg (cfu / mL); the μ value in the microdroplet culture system was... max It is 0.8183±0.01h -1 μ under traditional culture system maxIt is 0.807±0.08h -1 The λ value in the microdroplet culture system was 3.616±0.22 h, while that in the conventional culture system was 4.384±0.33 h; the T value in the microdroplet culture system was... 95 The time to T was 13.79 ± 0.65 h, under the traditional culture system. 95 It was 18.35 ± 1.64 h.

[0017] Invention effects: 1. The method provided in this application can be based on the relationship between the microbial culture performance of the target microorganism in a microdroplet culture system and a traditional culture system. During the microdroplet culture of microorganisms, the growth data of microorganisms under existing traditional culture systems (maximum bacterial concentration lg value (A), time to reach A (T)) can be used as a reference. 95 ), maximum specific growth rate (μ max This allows for more accurate prediction of microbial culture in microdroplet systems by incorporating parameters such as hysteresis time (λ). It can also be used to calibrate and validate the accuracy of commonly used optical methods (OD measurement) in microdroplet culture systems, establish a precise correspondence between OD values ​​and CFU, improve the data quality of existing platforms, and, through growth curve fitting, compare the differences and advantages / disadvantages between different methods.

[0018] 2. Achieve absolute quantification and accurate and reliable results: This application obtains absolute quantitative growth data by directly counting microbial colonies (CFU), which fundamentally avoids the inherent defects of OD value as an indirect indicator, and the results are not affected by factors such as strain characteristics and culture medium composition.

[0019] 3. High sensitivity, capable of monitoring the complete growth cycle: The detection sensitivity of this application can reach the single-cell level, which can accurately capture and quantify the growth dynamics of microorganisms in the lag phase and early exponential growth phase, and obtain a complete growth curve.

[0020] 4. Wide applicability and flexible operation: This application does not rely on specific optical detection equipment and is applicable to various types of microorganisms (bacteria, yeast, mold, etc.) and culture systems. It can be used as a general-purpose, high-precision analytical tool. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the growth curves of Escherichia coli in two systems in Example 1.

[0022] Figure 2 This is a schematic diagram of the comparison of modified Gompertz fitting parameters in Example 1.

[0023] Figure 3 This is a schematic diagram of the growth curves of Bacillus subtilis in two systems in Example 2.

[0024] Figure 4 This is a schematic diagram of the comparison of modified Gompertz fitting parameters in Example 2. Detailed Implementation

[0025] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0026] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. The terms "comprising" or "including" as used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to". The following descriptions are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0027] To address the problems of inaccurate and incomplete monitoring results when using optical measurement methods to monitor microbial growth curves in microdroplet culture systems in existing technologies, this application provides a method for obtaining the culture performance of target microorganisms in a microdroplet system, comprising: The target microorganism is activated and diluted to obtain the target bacterial solution; the target bacterial solution is cultured using a microdroplet culture system; the culture performance of the target microorganism in the microdroplet system is obtained based on the relationship between the culture performance of the target microorganism in the microdroplet system and the traditional culture system.

[0028] In this application, the culture performance refers to the growth of the target microorganism in a microdroplet culture system or a traditional culture system. It is a curve plotted with culture time on the x-axis and cell count or concentration on the y-axis, used to describe the entire dynamic process of microbial population growth, reproduction, and death. The culture performance described in this application includes the maximum cell concentration lg value (A) of the target microorganism during the culture process and the time (T) to reach A. 95 ), maximum specific growth rate (μ max ), delay time (λ).

[0029] In this application, the microdroplet culture system belongs to the microscale culture system, which refers to microscale droplets as independent culture units, in which microorganisms and culture medium are encapsulated in tiny droplets to form a closed, isolated, and uniform microculture environment, which can realize independent culture and high-throughput screening at the single-cell level, and belongs to the microscale, monoclonal level culture method.

[0030] In this application, the volume of microdroplets in the microdroplet culture system generally ranges from picoliters (pL) to microliters (μL), typically from 1 pL to 10 μL. This application does not limit the equipment or instruments used in the microdroplet culture system; those skilled in the art can select them based on known methods. Any system capable of generating, manipulating, and controlling microdroplets encapsulated in an immiscible carrier fluid, and culturing microorganisms within these microdroplets, falls within the scope of the microdroplet culture system described in this application.

[0031] In this application, the traditional culture system refers to a macro-scale conventional culture system, which uses conventional containers (petition dishes, shake flasks, culture bottles, test tubes, well plates, fermenters, etc.) as carriers to realize the culture of microbial communities in macro-volume culture media. The culture system is open or semi-open, and microorganisms are prone to material exchange, competition and symbiosis. It belongs to a batch-type, population-level culture method.

[0032] In this application, the traditional culture system mainly relies on macroscopic containers for culture. Compared with the microdroplet culture system mentioned above, the traditional culture system has a larger culture volume, generally ranging from milliliters (mL) to liters (L), usually ≥1mL.

[0033] In this application, the relationship between the culture performance of the target microorganism in the microdroplet system and the conventional culture system is obtained through the following steps: the target microorganism is activated and diluted to prepare a target bacterial solution; the target bacterial solution is cultured in both the microdroplet culture system and the conventional culture system; at a given time, the concentration (cfu / mL) of the target microorganism in the microdroplet culture system and the conventional culture system is obtained; based on the lg value of the concentration at the given time, the relationship between the culture performance of the target microorganism in the microdroplet culture system and the conventional culture system is obtained using the Gompertz model.

[0034] In this application, the steps for activating and diluting the target microorganism are not limited, and those skilled in the art can operate according to known methods. For example, the target microorganism can be dissolved first, then cultured for a certain period of time, and then serially diluted using sterile sodium chloride solution to obtain the target bacterial solution.

[0035] In this application, no limitation is made on the culture conditions for culturing the target bacterial solution using a microdroplet culture system or a traditional culture system. Those skilled in the art can select a suitable culture medium for culture based on the growth characteristics of the target microorganism and the requirements of the culture conditions, such as TSA medium, LB medium, enzyme medium, PDA medium, Schaeffer medium, TSB medium, DSM medium, etc. In addition to the selection of the culture medium, the setting of parameters such as temperature, oxygen, pH value, and aeration rate during the culture process can also be selected based on the growth characteristics and metabolic characteristics of the target microorganism, as long as the growth requirements of the target microorganism can be met.

[0036] In this application, the given time includes: 0h, 2h, 3h, 4h, 6h, 8h, 23h, and 24h.

[0037] In this application, obtaining the concentration of the target microorganism in the microdroplet culture system includes the following steps: collecting microdroplets at the given time; destroying the microdroplet structure to obtain a sample containing microorganisms; transferring the sample to a culture medium for cultivation to obtain the concentration of the target microorganism in the microdroplet culture system at the given time.

[0038] In some preferred embodiments, collecting microdroplets includes the following steps: introducing microdroplets into a culture tube, cutting off a tube segment containing microdroplets, and collecting the microdroplets in the tube segment, wherein the tube segment is 1-3cm, for example 1cm, 1.5cm, 2cm, 2.5cm, 3cm, preferably 2cm.

[0039] In this application, the culture tube may be selected as a culture tube with good gas permeability, preferably a Teflon tube.

[0040] In this application, after collecting microdroplets, the structure of the microdroplets is destroyed using a demulsifier, followed by filtration to obtain a sample containing microorganisms. The demulsifier is selected from one or more of Tween 80, Tween 65, Span 80, Span 60 or Span 20, or one or more of Tween 80, Tween 65, Span 80, Span 60 or Span 20 combined with lecithin, preferably Tween 80.

[0041] In this application, after obtaining a sample containing microorganisms, the sample is transferred to a culture medium for incubation to obtain the bacterial concentration (cfu / mL) at the current time point.

[0042] In this application, no limitation is made on the culture medium and culture conditions used to transfer the sample to the culture medium for culture. Those skilled in the art can select a suitable culture medium for culture according to the growth characteristics and culture conditions required by the target microorganism, such as TSA medium, LB medium, enzyme medium, PDA medium, Schaeffer medium, TSB medium, DSM medium, etc. In addition to the selection of culture medium, the setting of parameters such as temperature, oxygen, pH value, and aeration rate during the culture process can also be selected according to the growth characteristics and metabolic characteristics of the target microorganism, as long as the growth requirements of the target microorganism can be met.

[0043] In this application, no limitation is made on the method for obtaining the bacterial concentration. Those skilled in the art can operate according to known methods, such as the most commonly used spot counting of colonies (plate coating method) and dilution coating method.

[0044] In this application, the method for obtaining the concentration of the target microorganism in a conventional culture system differs from the method for obtaining the concentration in the microdroplet culture system described above, only in that the conventional culture system does not involve the process of collecting microdroplets, destroying the microdroplet structure, or filtering to obtain a sample containing microorganisms. The conventional culture system directly obtains the bacterial concentration (cfu / mL) at a given time point, for example, by using a point-counting method.

[0045] In this application, based on the Lg value of the target microorganism's concentration in a microdroplet culture system or a conventional culture system at a given time, the relationship between the cultivation performance of the target microorganism in the microdroplet culture system and the conventional culture system is obtained using the Gompertz model.

[0046] In this application, the Gompertz model uses the following formula to obtain the relationship between the culture performance of the target microorganism in the microdroplet culture system and the conventional culture system: (Formula 1) (Formula 2) In formula 1 or formula 2, Y(t) represents the logarithmic value of the target microbial concentration at time t in a microdroplet culture system or a traditional culture system, with base 10 [lg(cfu / mL)]. Y0 represents the logarithmic value of the initial concentration of the target microorganism in a microdroplet culture system or a traditional culture system, with the base 10 [lg(cfu / mL)]. A represents the logarithmic value of the maximum concentration of the target microorganism in a microdroplet culture system or a traditional culture system, with the base 10 [lg(cfu / mL)]. μ maxThis indicates the maximum specific growth rate (h) of the target microorganism in a microdroplet culture system or a conventional culture system. -1 ); λ represents the lag time (h) of the target microorganism in the microdroplet culture system or the traditional culture system. T 95 This indicates the time (h) it takes for the target microorganism to reach A in a microdroplet culture system or a traditional culture system.

[0047] In this application, A is also referred to as the platform value, and T is also referred to as the platform value. 95 Also known as the time to reach the plateau period.

[0048] In this application, the target microorganism includes bacteria, fungi, etc. The bacteria include Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, etc. The fungi include molds, yeasts, etc.

[0049] In some preferred embodiments, the target microorganism is *Escherichia coli*, and the alumina (A) of *E. coli* in the microdroplet culture system differs significantly from that in the conventional culture system, with the A in the microdroplet culture system being 0.5-1 [lg(cfu / mL)] greater than that in the conventional culture system; the μ in the microdroplet culture system... max Compared with μ in traditional culture systems max No significant difference; λ in the microdroplet culture system was not significantly different from that in the conventional culture system; T in the microdroplet culture system 95 Compared with the T in the traditional cultivation system 95 There were significant differences in T in the microdroplet culture system. 95 Compared to the traditional training system, T 95 Arrive 6-8 hours in advance.

[0050] In some preferred embodiments, the A value in the Escherichia coli microdroplet culture system is 9.779 ± 0.05 lg (cfu / mL), and the A value in the conventional culture system is 9.056 ± 0.16 lg (cfu / mL); the μ value in the microdroplet culture system... max It is 0.8917±0.03h -1 μ under traditional culture system max It is 0.8616±0.08h -1 The λ value in the microdroplet culture system was 1.288±0.09 h, while that in the conventional culture system was 0.7663±0.42 h; the T value in the microdroplet culture system was... 95 The time to T was 11.18 ± 0.20 h under the traditional culture system. 95It was 17.96 ± 1.80 h.

[0051] In some preferred embodiments, the target microorganism is Bacillus subtilis, and the aA of Bacillus subtilis in the microdroplet culture system differs significantly from that in the conventional culture system, with the aA in the microdroplet culture system being 1-3 [lg(cfu / mL)] greater than that in the conventional culture system; the μA in the microdroplet culture system... max Compared with μ in traditional culture systems max No significant difference was observed; however, the λ in the microdroplet culture system differed significantly from that in the conventional culture system, with the λ in the microdroplet culture system being 0.5-1 h shorter than that in the conventional culture system; the T in the microdroplet culture system... 95 Compared with the T in the traditional cultivation system 95 There were significant differences in T in the microdroplet culture system. 95 For: T under the traditional training system 95 Please arrive 4-6 hours in advance.

[0052] In some preferred embodiments, the A value of Bacillus subtilis in the microdroplet culture system is 9.131 ± 0.11 lg (cfu / mL), and the A value in the conventional culture system is 7.12 ± 0.03 lg (cfu / mL); the μ value in the microdroplet culture system... max It is 0.8183±0.01h -1 μ under traditional culture system max It is 0.807±0.08h -1 The λ value in the microdroplet culture system was 3.616±0.22 h, while that in the conventional culture system was 4.384±0.33 h; the T value in the microdroplet culture system was... 95 The time to T was 13.79 ± 0.65 h, under the traditional culture system. 95 It was 18.35 ± 1.64 h.

[0053] This application provides a method for obtaining the culture performance of target microorganisms in a microdroplet system. This method can be used to calibrate and verify the accuracy of commonly used optical methods (OD measurement) in microdroplet culture systems, establish a precise correspondence between OD values ​​and CFU, improve the data quality of existing platforms, and compare the differences and advantages and disadvantages between different methods through growth curve fitting.

[0054] Example 1: Comparison of Escherichia coli culture performance using microdroplet culture method and conventional culture method 1.1 Materials and Equipment Equipment: High-throughput micro-level droplet culture omics system (MISS Cell, Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.); biosafety cabinet; membrane filter microbial collector; incubator; vortex apparatus.

[0055] Materials: Escherichia coli CMCC(B)44102 (China Center for Medical Bacteriological Culture Collection, commercially available); tryptic soy peptone broth (TSB); sample vials; Teflon coils; droplet-generating oil; sterile filter cups (0.45 μm); 0.9% sterile sodium chloride solution; sterile sodium chloride peptone buffer at pH 7.0 containing 4% Tween 80; pre-prepared tryptic soy peptone agar plates (TSA).

[0056] 1.2 Experimental Methods 1.2.1 Preparation of bacterial culture Strain activation: Take one vial of Escherichia coli lyophilized powder, dissolve it thoroughly in 1 mL of TSB medium, add 100 μL of the dissolved bacterial solution to a sterile test tube containing 10 mL of TSB medium, and incubate at 30-35℃ for 18-24 hours.

[0057] Bacterial culture dilution: The above culture was serially diluted with 0.9% sterile sodium chloride solution to obtain 10... 5 cfu / mL (10) -4 (Dilution grade), 10 3 cfu / mL (10) -6 (Dilution level) bacterial solution.

[0058] Bacterial count: Take 10 3 cfu / mL (10) -6 Add 100 μL of diluted bacterial suspension to a filter cup, rinse with 100 mL of PB containing 4% Tween 80, place the filter membrane on a TSA plate, set up two replicates for each group, incubate at 30~35℃, record the number after a period of time, and observe the colony growth.

[0059] 1.2.2 Sample Preparation and Cultivation Microdroplet system: Take 10 5 Add 300 μL of cfu / mL bacterial culture to 30 mL of TSB in a sample vial and mix thoroughly. Follow the standard operating procedure for the MISS Cell system to complete sample injection and droplet generation. Seal both ends of the tube and incubate at 30–35°C.

[0060] Traditional cultivation method: Take 10 3 Mix 100 μl of cfu / mL bacterial culture with 500 mL of TSB, mix thoroughly, dispense into test tubes (10 mL / tube), and incubate at 30–35 °C.

[0061] 1.2.3 Sampling Count Sampling and counting were performed at each time point: 0h, 2h, 3h, 4h, 6h, 8h, 23h, and 24h for both the microdroplet system and the traditional culture method.

[0062] Microdroplet system: Using sterile scissors, cut a 2cm segment (containing approximately 10-15 droplets) from the culture tube. Seal both ends of the remaining section with clamps and continue incubation at 30-35°C. Allow the droplets to flow out by gravity from the cut segment, placing each droplet in a sterile 1.5mL centrifuge tube. Take four droplets at each time point as parallels. Add 1mL of sterile pH 7.0 sodium chloride peptone buffer containing 4% Tween 80 to each centrifuge tube containing droplets, mix thoroughly by pipetting, and dilute 10-fold with 0.9% sodium chloride solution to a suitable concentration for counting. Take 1 mL of each dilution and filter it using the membrane filtration method. After filtration, rinse with 100 mL of PB containing 4% Tween 80. Carefully remove the filter membrane with sterile forceps, colony side up, and affix it to a TSA plate. Incubate at 30-35℃ for 24 hours and count the number of colonies. Select dilutions with a colony count of no more than 250 CFU and convert them to the bacterial concentration (CFU / mL) at the current time point.

[0063] Traditional culture method: Take 4 test tubes as parallels at each time point and dilute them 10-fold with 0.9% sodium chloride solution to a suitable counting concentration. Take 1 mL of each dilution and filter it using the membrane filtration method. After filtration, rinse with 100 mL of PB containing 4% Tween 80. Carefully remove the filter membrane with sterile forceps, colony side up, and affix it to a TSA plate. Incubate at 30-35℃ for 24 hours, then count the colonies. Select the dilution with a colony count not exceeding 250 CFU and convert it to the bacterial concentration (CFU / mL) at the current time point.

[0064] 1.2.4 Growth Curve Plotting and Model Fitting The microbial concentration data calculated at each time point were compiled into a table. With the culture time as the horizontal axis and the lg value of microbial concentration (lg / mL) as the vertical axis, an absolute quantitative growth curve of Escherichia coli in the droplet was plotted.

[0065] The Gompertz first-order model is a function specifically designed to describe microbial growth, and its coefficient of determination R0 is... 2 The correlation of the experimental fitting parameters is described. The closer the coefficient is to 1, the higher the goodness of fit and the better the correlation between the parameters. The first-order model describes the relationship between time and biomass [lg(cfu / mL)]. The fitting parameters of the Gompertz model are corrected using GraphPad Prism (Equation 1).

[0066] (Formula 1) Y( t) represents the logarithmic value of the microbial concentration at time t, base 10 [lg(cfu / mL)]; Y0 represents the logarithmic value of the initial concentration, base 10 [lg(cfu / mL)]; A represents the logarithmic value of the maximum concentration, base 10 [lg(cfu / mL)]; μ max Represents the maximum specific growth rate (h) -1 ); λ represents the delay time (h).

[0067] 1.3 Results and Analysis The changes in bacterial concentration over time after conversion from experimental counts are shown in Table 1. The results of correcting the Gompertz fitting parameters are shown in Table 2.

[0068] Table 1. lg values ​​of bacterial concentration (cfu / mL) results

[0069] Table 2. Corrected Gompertz Fitting Parameters

[0070] As can be seen from Table 2, R 2 All values ​​are greater than 0.998, indicating that the modified Gompertz equation has good correlation. The fitting results are as follows: Figure 1 As shown, the values ​​of A for the conventional group and the microdroplet group were 9.056 lg (cfu / mL) and 9.779 lg (cfu / mL), respectively; μ max 0.8616h respectively -1 0.8917h -1 The time λ was 0.7663 h and 1.288 h, respectively. The t-test results (Graphpad Prism) showed no significant difference in λ between the traditional group and the microdroplet group. This application used the Gompertz model to calculate the time T to reach A. 95 Further describing the differences in growth kinetics between the microdroplet group and the conventional group (Equation 2), the average T of the conventional group was calculated. 95 The average T for the microdroplet group was approximately 17.96 h. 95 It takes approximately 11.18 hours, and the microdroplet group is expected to reach the plateau phase about 7 hours earlier.

[0071] (Formula 2) The calculations yielded A and μ values ​​for the traditional group and the microdroplet group. max , λ, T 95 Statistical analysis was performed, and the results are as follows: Figure 2 As shown, the t-test results indicate that the A and T values ​​of the traditional group and the microdroplet group are significantly different. 95 There was a significant difference (P<0.05); μ maxThere was no significant difference between λ and λ.

[0072] In summary, this method can accurately determine the growth curve of Escherichia coli within microdroplets, and by combining it with the Gompertz model, the differences between this method and traditional culture methods can be quantitatively compared.

[0073] Example 2: Comparison of the culture performance of Bacillus subtilis using microdroplet culture method and conventional culture method 2.1 Equipment and Materials Equipment: High-throughput micro-level droplet culture omics system (MISS Cell, Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.); biosafety cabinet; membrane filter microbial collector; incubator; vortex apparatus.

[0074] Materials: Bacillus subtilis CMCC(B)63501 (China Center for Medical Bacteriological Culture Collection, commercially available); tryptic soy peptone broth (TSB); sample vials; Teflon tubing; droplet-generating oil; sterile filter cups (0.45 μm); 0.9% sterile sodium chloride solution; pH 7.0 sterile sodium chloride peptone buffer containing 4% Tween 80; pre-prepared tryptic soy peptone agar plates (TSA).

[0075] 2.2 Experimental Methods 2.2.1 Preparation of bacterial culture Strain activation: Take one vial of Bacillus subtilis lyophilized powder, dissolve it thoroughly in 1 mL of TSB medium, add 100 μL of the dissolved bacterial solution to a sterile test tube containing 10 mL of TSB medium, and incubate at 30-35℃ for 18-24 hours.

[0076] Bacterial culture dilution: The above culture was serially diluted with 0.9% sterile sodium chloride solution to obtain 10... 5 cfu / mL (10) -2 (Dilution grade), 10 3 cfu / mL (10) -4 (Dilution level) bacterial solution.

[0077] Bacterial count: Take 10 3 cfu / mL (10) -4 Add 100 μL of diluted bacterial suspension to a filter cup, rinse with 100 mL of PB containing 4% Tween 80, place the filter membrane on a TSA plate, set up two replicates for each group, incubate at 30~35℃, record the number after a period of time, and observe the colony growth.

[0078] 2.2.2 Sample Preparation and Cultivation The preparation and cultivation process is the same as 1.2.2 in Example 1 above.

[0079] 2.2.3 Sampling Count The counting process is the same as 1.2.3 in Example 1 above.

[0080] 2.2.4 Growth Curve Plotting and Model Fitting The plotting and fitting process is the same as 1.2.4 in Example 1 above.

[0081] 2.3 Results and Analysis The changes in bacterial concentration over time after conversion from experimental count results are shown in Table 3. The results of correcting the Gompertz fitting parameters are shown in Table 4.

[0082] Table 3. lg values ​​of bacterial concentration (cfu / mL) results

[0083] Table 4. Corrected Gompertz Fitting Parameters

[0084] As can be seen from Table 4, R 2 All values ​​are greater than 0.99, indicating that the modified Gompertz equation has good correlation. The fitting results are as follows: Figure 3 As shown, the values ​​of A for the conventional group and the microdroplet group were 7.12 lg (cfu / mL) and 9.131 lg (cfu / mL), respectively; μ max 0.8070h respectively -1 0.8183h -1 The time λ was 4.384 h and 3.616 h, respectively. The t-test results (Graphpad Prism) showed significant differences in A and λ between the traditional group and the microdroplet group (P < 0.05). This application used the Gompertz model to calculate the time T to reach A. 95 The differences in growth kinetics between the microdroplet group and the conventional group of Bacillus subtilis were further described (Equation 2), and the average T of the conventional group was calculated. 95 The average T for the microdroplet group was approximately 18.35 h. 95 It takes approximately 13.79 hours, and the microdroplet group is expected to reach the plateau phase about 5 hours earlier.

[0085] The calculations yielded A and μ values ​​for the traditional group and the microdroplet group. max , λ, T 95 Statistical analysis was performed, and the results are as follows: Figure 4 As shown, the t-test results indicate that the A and T values ​​of the traditional group and the microdroplet group are significantly different. 95 There are significant differences between μ and λ; max No significant difference.

[0086] In summary, this method can accurately determine the growth curve of Bacillus subtilis within microdroplets, and by combining it with the Gompertz model, the differences between this method and traditional culture methods can be quantitatively compared.

[0087] The description in this disclosure is provided for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the disclosure to its forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of this disclosure and to enable those skilled in the art to understand this disclosure and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for obtaining the culture performance of a target microorganism in a microdroplet culture system, comprising: The target microorganism is activated and diluted to obtain the target bacterial solution; The target bacterial culture was cultured using a microdroplet culture system; Based on the relationship between the culture performance of the target microorganism in the microdroplet culture system and the traditional culture system, the culture performance of the target microorganism in the microdroplet culture system was obtained.

2. The method according to claim 1, wherein, The culture performance includes the maximum bacterial concentration (A) and the time to reach A (T). 95 ), maximum specific growth rate (μ max ), delay time (λ).

3. The method according to claim 1, wherein, The relationship between the culture performance of the target microorganism in the microdroplet culture system and the conventional culture system was obtained through the following steps: The target microorganism is activated and diluted to obtain the target bacterial solution; The target bacterial culture was cultured using both a microdroplet culture system and a traditional culture system. At a given time, the concentration (cfu / mL) of the target microorganism in the microdroplet culture system and the conventional culture system was obtained. Based on the lg value of the concentration at the given time, the relationship between the culture performance of the target microorganism in the microdroplet culture system and the traditional culture system was obtained using the Gompertz model.

4. The method according to claim 3, wherein, The given time includes: 0h, 2h, 3h, 4h, 6h, 8h, 23h, and 24h.

5. The method according to claim 3, wherein, Obtaining the concentration of the target microorganism in the microdroplet culture system includes the following steps: Collect microdroplets at the given time. The microdroplet structure was disrupted to obtain a sample containing the target microorganism; The above samples were transferred to a culture medium for incubation to obtain the concentration of the target microorganism in the microdroplet culture system at the given time.

6. The method according to claim 5, wherein, The process of collecting microdroplets includes: introducing microdroplets into a culture tube, cutting off a tube segment containing microdroplets, and collecting the microdroplets within the tube segment. The pipe section is 1-3cm, preferably 2cm.

7. The method according to claim 5, wherein, Obtaining a sample containing the target microorganism involves: disrupting the microdroplet structure using a demulsifier, followed by filtration to obtain the sample containing the target microorganism. The demulsifier is selected from one or more of Tween 80, Tween 65, Span 80, Span 60 or Span 20, or one or more of Tween 80, Tween 65, Span 80, Span 60 or Span 20 combined with lecithin.

8. The method according to claim 3, wherein, The Gompertz model uses the following formula to obtain the relationship between the culture performance of the target microorganism in the microdroplet culture system and the conventional culture system: (Official 1) (Official 2) In formula 1 or formula 2, Y(t) represents the logarithmic value of the target microbial concentration at time t in a microdroplet culture system or a traditional culture system, with base 10 [lg(cfu / mL)]. Y0 represents the logarithmic value of the initial concentration of the target microorganism in a microdroplet culture system or a traditional culture system, with the base 10 [lg(cfu / mL)]. A represents the logarithmic value of the maximum concentration of the target microorganism in a microdroplet culture system or a traditional culture system, with the base 10 [lg(cfu / mL)]. μ max This indicates the maximum specific growth rate (h) of the target microorganism in a microdroplet culture system or a conventional culture system. -1 ); λ represents the lag time (h) of the target microorganism in the microdroplet culture system or the traditional culture system. T 95 This indicates the time (h) for the target microorganism to reach A in a microdroplet culture system or a traditional culture system.

9. The method according to any one of claims 1-8, wherein, The target microorganism is Escherichia coli. The A value of *Escherichia coli* in the microdroplet culture system differed significantly from that in the conventional culture system; μ in the microdroplet culture system max Compared with μ in traditional culture systems max No significant difference; λ in the microdroplet culture system was not significantly different from that in the conventional culture system; T in the microdroplet culture system 95 Compared with the T in the traditional cultivation system 95 There are significant differences; Alternatively, the target microorganism may be Bacillus subtilis. The A value of Bacillus subtilis in the microdroplet culture system differed significantly from that in the conventional culture system; μ in the microdroplet culture system max Compared with μ in traditional culture systems max No significant difference; λ in the microdroplet culture system was significantly different from that in the traditional culture system; T in the microdroplet culture system 95 Compared with the T in the traditional cultivation system 95 There are significant differences.

10. The method according to claim 9, wherein, The A value of Escherichia coli in the microdroplet culture system is 0.5-1 lg (cfu / mL) greater than that in the conventional culture system. The Escherichia coli in the microdroplet culture system T 95 For: T under the traditional training system 95 Arrive 6-8 hours in advance; The A value of Bacillus subtilis in the microdroplet culture system is 1-3 lg (cfu / mL) greater than that in the conventional culture system. The λ of Bacillus subtilis in the microdroplet culture system is 0.5-1 h shorter than that in the conventional culture system; The Bacillus subtilis in the microdroplet culture system T 95 For: T under the traditional training system 95 4-6 hours in advance Preferably, The A value in the Escherichia coli microdroplet culture system was 9.779 ± 0.05 lg (cfu / mL), while the A value in the conventional culture system was 9.056 ± 0.16 lg (cfu / mL); the μ value in the microdroplet culture system was... max It is 0.8917±0.03h -1 μ under traditional culture system max It is 0.8616±0.08h -1 The λ value in the microdroplet culture system was 1.288±0.09 h, while that in the conventional culture system was 0.7663±0.42 h; the T value in the microdroplet culture system was... 95 The time to T was 11.18 ± 0.20 h under the traditional culture system. 95 It was 17.96 ± 1.80 h; The A value of *Bacillus subtilis* in the microdroplet culture system was 9.131 ± 0.11 lg (cfu / mL), while the A value in the conventional culture system was 7.12 ± 0.03 lg (cfu / mL); the μ value in the microdroplet culture system was... max It is 0.8183±0.01h -1 μ under traditional culture system max It is 0.807±0.08h -1 The λ value in the microdroplet culture system was 3.616±0.22 h, while that in the conventional culture system was 4.384±0.33 h; the T value in the microdroplet culture system was... 95 The time to T was 13.79 ± 0.65 h, under the traditional culture system. 95 It was 18.35 ± 1.64 h.

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