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Summary

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Abstract

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Introduction

Microbial cultivation is an important foundation for microbiological scientific research and industrial applications, which is widely used in the isolation, identification, reconstruction, screening, and evolution of microorganisms^1,2,3. Conventional microbial cultivation methods mainly use test tubes, shake flasks, and solid plates as cultivation containers, combined with shaking incubators, spectrophotometers, microplate readers, and other equipment for microbial cultivation, detection, and screening. However, these methods have many problems, such as cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. The high-throughput cultivation methods developed in recent years are mainly based on the microplate. But the microplate has a low level of dissolved oxygen, poor mixing property, and severe evaporation and thermal effect, which often lead to poor growth status and experiment parallelization of microorganisms^4,5,6,7; on the other hand, it needs to be equipped with expensive equipment, such as liquid-handling workstations and microplate readers, to achieve automated cultivation and process detection^8,9.

As an important branch of microfluidic technology, droplet microfluidics has been developed in recent years based on traditional continuous-flow microfluidic systems. It is a discrete flow microfluidic technology that uses two immiscible liquid phases (usually oil-water) to generate dispersed micro-droplets and operate on them¹⁰. Because micro-droplets have the characteristics of small volume, large specific surface area, high internal mass transfer rate, and no cross-contamination caused by compartmentalization, and the advantages of strong controllability and high throughput of droplets, there have been many kinds of research applying droplet microfluidic technology in high-throughput cultivation, screening, and evolution of microorganisms¹¹. However, there are still a series of key issues to make droplet microfluidic technology popularized and widely applied. Firstly, the operation of droplet microfluidics is cumbersome and intricate, resulting in high technical requirements for operators. Secondly, droplet microfluidic technology combines optical, mechanical, and electrical components and needs to be associated with biotechnology application scenarios. It is difficult for a single laboratory or team to build efficient droplet microfluidic control systems if there is no multi-disciplinary collaboration. Thirdly, on account of the small volume of micro-droplet (from picoliter (pL) to microliter (μL)), it takes much difficulty to realize the precise automated control and real-time online detection of droplets for some basic microbial operations such as sub-cultivation, sorting, and sampling, and it is also difficult to construct an integrated equipment system¹².

In order to address the above problems, an automatic Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology¹³. The MMC consists of four functional modules: a droplet recognition module, a droplet spectrum detection module, a microfluidic chip module, and a sampling module. Through the system integration and control of all the modules, automated operation system including the generation, cultivation, measurement (optical density (OD) and fluorescence), splitting, fusion, sorting of droplets is accurately established, achieving the integration of functions such as inoculation, cultivation, monitoring, sub-cultivation, sorting and sampling required by the process of microbial droplet cultivation. MMC can hold up to 200 replicate droplet cultivation units of 2-3 µL volume, which is equivalent to 200 shake flask cultivation units. The micro-droplet cultivation system can satisfy the requirements of non-contamination, dissolved oxygen, mixing, and mass-energy exchange during the growth of microorganisms, and meet the various needs of microbial research through multiple integrated functions, for instance, growth curve measurement, adaptive evolution, single factor multi-level analysis, and metabolite research and analysis (based on fluorescence detection)^13,14.

Here, the protocol introduces how to use the MMC to conduct automated and microbial cultivation and adaptive evolution in detail (Figure 1). We took wild-type Escherichia coli (E. coli) MG1655 as an example to demonstrate the growth curve measurement and a methanol-essential E. coli strain MeSV2.2¹⁵ to demonstrate the adaptive evolution in MMC. An operation software for MMC was developed, which makes the operation very simple and clear. In the whole process, the user needs to prepare the initial bacteria solution, set the conditions of the MMC, and then inject the bacteria solution and related reagents into the MMC. Subsequently, the MMC will automatically perform operations such as droplet generation, recognition and numbering, cultivation, and adaptive evolution. It also will perform online detection (OD and fluorescence) of the droplets with high time resolution and display the related data (which can be exported) in the software. The operator can stop the cultivation process at any time according to the results and extract the target droplets for subsequent experiments. The MMC is easy to operate, consumes less labor and reagents, and has relatively high experimental throughput and good data parallelity, which has significant advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and robust experimental platform for researchers to conduct related microbial research.

Protocol

1. Instrument and software installation

1. Choose a clean and sterile environment (such as a clean bench) as a dedicated permanent space for MMC. Install the MMC steadily in the space.
   NOTE: Keep the MMC away from the interference of strong electric fields, magnetic fields, and strong heat radiation sources. Avoid severe vibration from affecting the optical detection components. Provide the power supply of AC220 V, 50 HZ to the MMC. For details on MMC refer to the Table of Materials and the website for MMC¹⁶.
2. Install the operation software from the MMC.zip file
   NOTE: Contact the authors for the MMC.zip file.
   1. Create a dedicated folder and save the zip file in it.
   2. Create another dedicated folder as the "Installation Directory". Unzip the MMC.zip and save the files in the new folder.
      NOTE: The computer configuration is best to meet: (1) Windows 7 64-bit operating system or above; (2) CPU: i5 or above; (3) memory: 4 GB or above; (4) hard disk: 300 GB or above (rotational speed greater than 7200 rpm or solid-state disk).

2. Preparations

1. Connect the syringe needle (inner diameter is 0.41 mm and outer diameter is 0.71 mm), quick connector A, and reagent bottle (Figure 2C), and autoclave them at 121 °C for 15 min.
   NOTE: Unscrew the cap of the reagent bottle slightly during sterilization. A few more reagent bottles can be prepared each time for use.
2. Use a 0.22 µm polyvinylidene fluoride (PVDF) filter to filter MMC oil. Put the microfluidic chip (Figure 2B) and MMC oil into the clean bench in advance and sterilize them by ultraviolet irradiation for 30 min before use.
   NOTE: For the details of Quick connector A, reagent bottle, MMC oil and microfluidic chip refer to the Table of Materials.
3. Install the microfluidic chip
   1. Open the door of the operation chamber (Figure 2A) and lift the optical fiber probe.
   2. Align the electric field holes with the electric field needles and gently place the chip on the chip pedestal. Then insert the two positioning columns into the positioning holes and put down the optical fiber probe (Figure 2D).
   3. Connect the quick connector A on the chip to the corresponding port of the MMC according to the position number (C5-O5, C4-O4, C6-O6, C2-O2, CF-OF, C1-O1, C3-O3). Then close the door of the operation chamber.
4. Replenish the MMC oil (to about 80 mL) in the oil bottle and empty the waste liquid in the waste bottle before use.
   NOTE: The waste liquid is usually organic waste. Please refer to regional law and regulation upon disposal, subject to change based on experimental setup.

3. Growth curve measurement in MMC

1. Preparation for initial bacterial solution
   1. Follow the related standard regulations to prepare Luria-Bertani (LB) medium and autoclave at 121°C for 15 min.
      NOTE: Components of LB medium: NaCl (10 g/L), yeast extract (5 g/L) and tryptone (10 g/L).
   2. Take out the E. coli MG1655 strain from glycerol stock and cultivate it in a 50 mL shake flask with 10 mL of LB medium in a shaking incubator (200 rpm) at 37 °C for 5-8 h.
      NOTE: The cultivation time depends on the specific strains. It is optimal to cultivate the strain to the logarithmic period/phase.
   3. Dilute the cultured E. coli MG1655 solution with fresh medium to an OD₆₀₀ of 0.05-0.1 to obtain an initial bacteria solution (prepare about 10 mL).
2. Click on Initialization to initialize the MMC. After the initialization interface appears, set the cultivation temperature as 37 °C and the photoelectric signal value as 0.6 (Figure 3A). Initialization will take about 20 min.
3. Turn on the UV lamp (wavelength 254 nm) during initialization.
4. Inject the initial bacteria solution and MMC oil into the reagent bottle.
   1. Take out a sterilized reagent bottle on the clean bench and tighten the cap.
   2. Use a 10 mL sterile syringe to inject 3-5 mL of MMC oil from the syringe needle of the side tube. Tilt and rotate the reagent bottle slowly to make the oil fully infiltrate the inner wall.
   3. Inject about 5 mL of initial bacteria solution, and then fill the reagent bottle by injecting 5-7 mL of the oil again.
   4. Pull out the independent quick connector A, and insert the quick connector A of the reagent bottle into its quick connector B to complete the sample injection operation (Figure 4A).
5. Wait for the initialization to end and then turn off the UV lamp (wavelength 254 nm).
6. Open the door of the operation chamber, and put the reagent bottle into the metal bath.
7. Pull out the C2 connector of the chip and the quick connector A of the reagent bottle. Connect the side tube connector of the reagent bottle to the C2 connector and the top tube connector to the O2 connector. Then close the door of the operation chamber.
8. Click on Growth Curve to choose the function of growth curve measurement (Figure 3A). In the parameter setting interface, input the Number as 15, turn on the OD detection switch and set the Wavelength as 600 nm. Click on Start to start droplet generation. It will take about 10 min.
   NOTE: Here, Number refers to the number of droplets to be generated. Wavelength refers to the wavelength of the OD to be detected. Set the Number (maximum 200) and Wavelength (350-800 nm) according to the experiment requirements.
9. When a pop-up window appears on the main interface prompting "Remove the reagent bottle between C2 and O2, then please click the OK button after completion", open the door of the operation chamber to take out the reagent bottle and connect the C2 and O2 connectors.
10. Close the door, and click the OK button in the pop-up window to automatically cultivate the droplets and detect the OD values.
    NOTE: The MMC detects the OD value when the droplet passes the optical fiber probe. Therefore, the detection period depends on the number of droplets generated.
11. When the growth curve reaches the stationary phase, click the Data Export button to export the OD data. Select the data save path and export the OD value recorded during the cultivation period in the .csv format, which can be opened by appropriate software (e.g., Microsoft Excel). Then use a mapping software (e.g., EXCEL and Origin 9.0) to plot the growth curve.
    NOTE: During the cultivation process, it is feasible to click on the Data Export at any time to export the OD data of all the current droplets.

4. Adaptive evolution in MMC

1. Preparation for initial bacterial solution
   1. Follow the related standard regulations to prepare the special liquid medium and solid plates for the MeSV2.2 and autoclave at 121 °C for 15 min.
      NOTE: For the components of the special medium refer to Table 1 and the Table of Materials.
   2. Cultivate the MeSV2.2 using the solid plate (diameter = 90 mm) in a 37 °C constant temperature incubator for 72 h. Then pick an independent colony and cultivate it in a 50 mL shake flask with 10 mL of the special liquid medium in a shaking incubator (200 rpm) at 37 °C for 72 h.
   3. Dilute the cultured MeSV2.2 solution with the medium to an OD₆₀₀ of 0.1-0.2 (ensure that the total volume is not less than 10 mL) and continue cultivating it in the shake flask for 5 h to obtain the initial bacteria solution.
      NOTE: The MeSV2.2 is a methanol-essential E. coli strain. The special liquid medium contains 500 mmol/L methanol, which is a strong stress for MeSV2.2, resulting in very slow growth. Note that obtaining the initial bacteria solution here is different from that described in step 3.1.
2. Initialize the MMC as explained in steps 3.2, 3.3, and 3.5.
3. Take out two sterilized reagent bottles, one of which is for the initial bacteria solution and the other is for the fresh medium. Inject the initial bacteria solution (5 mL), fresh medium (12-15 mL), and MMC oil into the reagent bottles as explained in step 3.4.
   NOTE: As adaptive evolution is a long-term process involving multiple sub-cultivations, store as much fresh medium as possible in MMC. The medium cannot be replenished during the experiment running.
4. Install the two reagent bottles into MMC as explained in step 3.6. Install the one for the initial bacteria solution between the C2 and O2 connector and the other for the fresh medium between the C4 and O4 connector.
5. Click on ALE to choose the function of adaptive evolution (Figure 3B). In the parameter setting interface, turn on the OD Detection switch.
6. Set the Number as 50, Wavelength as 600 nm, Concentration as 0%, Type as Time, Parameter as 30 h, and Repetitions as 99. Click on Start to start droplet generation. It will take about 25 min.
   NOTE: Here, "Concentration" refers to the maximum concentration of chemical factors for adaptive evolution. For different droplets, it is realizable in MMC to introduce different concentrations of chemical factors to provide different growth conditions. Calculate the introduced concentrations using the following equation:
   
   Here "C" refers to the concentration of chemical factors introduced into droplets; "a" refers to the concentration of chemical factors in the reagent bottles between the C4 and O4 connector; "b" refers to the concentration of chemical factors in the reagent bottles between the C6 and O6 connector; and "i" refers to the available concentration. There are eight concentrations available in MMC. Since the chemical factor here has a single concentration (500 mmol/L methanol) and it is one of the ingredients of the medium, only one reagent bottle containing the chemical factor is installed here, and the Concentration is set as 0%. Type refers to the mode of sub-cultivation, which is divided into three types: time mode, OD value mode, and fluorescence mode. The former means to cultivate the droplets for a fixed time and then sub-cultivate, while the latter two means to cultivate the droplets to pre-defined OD value/fluorescence intensity and then sub-cultivate. Parameter refers to the related parameter required when choosing a mode of sub-cultivation. Repetitions refers to the number of sub-cultivations.
7. Remove the reagent bottle placed between the C2 and O2 connector as explained in step 3.8.
8. Observe whether the maximum OD values of the droplets during each sub-cultivation period have increased significantly. If the increase occurs and meets the experiment requirements, click on the Data Export button to export the OD data as explained in step 3.9.
   NOTE: Here, the sub-cultivation period depends on the Parameter. For example, when setting Type as Time and Parameter as 30 h, the sub-cultivation period is 30 h. During each sub-cultivation period, there are the maximum OD values of the droplets. Estimate whether the adaptive evolution meets the experiment requirements by the increase of maximum OD values (The increase depends on the actual cultivation process of the strain, for example, increased by more than 20%).
   CAUTION: Pay attention to whether the stored fresh medium is exhausted. If the significant increase has not occurred even after the medium is exhausted, extract the better-growing droplets and carry out a new round of adaptive evolution.
9. Extract the target droplets from the MMC.
   1. Click on the Screening button to choose the function of droplet extraction (Figure 3C). Choose the Collect option, click on the numbers of target droplets, and then click on OK.
      NOTE: Droplet screening includes "Collect", "Discard" and "Extract seed solution". "Extract seed solution" means to collect the remaining droplets¹³ after the sub-cultivation operation.
   2. Wait for the pop-up window to prompt, "Please pull out the CF quick connector and put it into the EP tube". Put the CF quick connector into the microcentrifuge tube for collection according to the software prompt and then click on OK (Figure 4D).
   3. After 1-2 min, the software interface will pop up a new window prompting, "Please insert the connector back and click OK if finished". Then, insert the CF quick connector back and click on OK to make MMC continue to run (Figure 4D). When the next target droplet reaches the droplet recognition site, repeat 4.9.2-4.9.3 to collect it.
      NOTE: After all the target droplets are collected, the MMC will continue cultivating the remaining droplets. If the cultivation is not necessary, click on Stop to directly terminate the operation.
   4. Extract the droplet using a 2.5 µL pipette, drop it on the 90 mm agarose plate, and spread it evenly with a triangular glass spreading rod with a side length of 3 cm. Then cultivate it in a 37 °C constant temperature incubator for 72 h.
   5. Pick 3-5 independent colonies and separately cultivate them in the 50 mL shake flasks with 10 mL of fresh medium in a shaking incubator (200 rpm) at 37 °C for 48-72 h. Follow the related standard regulations to store the cultured bacteria solution in the glycerol tube for subsequent experiments.

5. Clean of the MMC

1. After completion of the experiment, click on Stop to stop all the operations. Then click on Clean to clean the chip and tubes. It will take about 15 min.

Representative Results

This protocol uses E. coli MG1655 and a MeSV2.2 strain as examples to demonstrate the microbial cultivation and methanol-essential adaptive evolution with an automated and relatively high-throughputstrategy in MMC. The growth curve measurement was mainly used to characterize microbial cultivation. The adaptive evolution was conducted by automated continuous sub-cultivation and adding a high concentration of methanol as the selective pressure during each sub-cultivation. Whether adaptive evolution had been realized was estimated through the variation trend of the maximum OD value of the droplets during each sub-cultivation period. The tunable parameters and accuracy parameters of MMC are shown in Table 2.

Results of growth curve measurement
The OD₆₀₀ values of the 15 droplets detected during the cultivation process were exported from the MMC after cultivating for about 20 h (Figure 5A). It can be observed that the detection was conducted approximately every 14 min. This detection period depends on the number of droplets generated because the droplets are cycled back and forth in the tubes for cultivation, and the detection module only detects the OD values (the detection and calculation of OD value are shown in Supplementary Figure 1) when the droplets pass the optical fiber probe. Therefore, the 14 min is a very short detection period, providing a high time resolution detection process to reflect the growth of microorganisms more accurately.

According to the exported data, the average OD₆₀₀ values and standard deviation (SD) of 15 droplets at each time point were calculated, and the growth curve of E. coli MG1655 was plotted (Figure 5B). The results show that the growth curve presents an "S" shape, including lag phase, logarithmic phase, and stationary phase, which is very consistent with the classic microbial growth model. At the same time, the standard deviations of 15 droplets are very small, indicating good growth consistency and parallelity. Thus, it fully demonstrates the good microbial cultivation and detection performance of MMC. Moreover, it was also verified that there is little crosstalk between droplets during cultivation (Supplementary Figure 2 and Supplementary Table 1).

Results of adaptive evolution
We have performed a long-term adaptive evolution of MeSV2.2 in MMC. On the 18^th day, according to the increasing trend of the maximum OD₆₀₀ values of the droplets during each sub-cultivation period from the growth curves displayed on the software interface, we believed that a good adaptive evolution was achieved in the 50 droplets. The OD₆₀₀ data was exported and 8 droplets (including droplet 6) with relatively good growth performance were extracted¹³. Figure 6A shows the growth curves of 50 droplets in the whole adaptive evolution process. In 18 days, MMC has automatically carried out 13 sub-cultivation operations. It can be seen from Figure 6A that MeSV2.2 grows slowly first and fast afterward, which indicates the track of adaptive evolution in MeSV2.2. To supply a selection pressure, the methanol was added to the MeSV2.2 medium. Initially, methanol inhibited cell growth. After the adaptive evolution, the enriched cells adapted to methanol had a higher growth rate. The growth curve of droplet 6 in the whole adaptive evolution process was plotted separately (Figure 6B). The maximum OD₆₀₀ values in the first generation and last sub-cultivation period were 0.37 and 0.58, respectively, increased by 56.8%. It indicates that the strain in droplet 6 has realized an obvious adaptive evolution.

Subsequently, droplet 6 strain and the initial strain in shake flasks were cultivated, and their growth curves were compared (Figure 6C). According to the methods given in the literature^17,18, the maximum specific growth rates (µ_max) of the droplet 6 strain and the initial strain were calculated, which were 0.096 h^-1 and 0.072 h^-1, respectively. Figure 6C reveals that the droplet 6 strain exhibited a higher maximum specific growth rate (increasing by 54.8%) and had a higher cell concentration in the stationary phase (increasing by 20.0%) than the initial strain when cultivated in shake flasks, which further suggested that the adaptive evolution in MeSV2.2 has been realized.

Figure 1: Overall workflow of growth curve measurement and adaptive evolution in MMC. (A) Growth curve measurement in MMC. Firstly, cultivate the strain in shake flask to prepare the initial bacterial solution. Then, inject the initial bacteria solution into the reagent bottle. Next, generate the droplets in MMC. MMC makes the droplets cycle back and forth in the microfluidic chip and tubes to cultivate them. When droplets pass the detection site, the OD data will be detected and recorded. Finally, export the data for analysis. (B) Adaptive evolution in MMC. Pick a single colony from the agarose plate and cultivate it in a shake flask to prepare the initial bacterial solution. After injecting the initial bacteria solution into the reagent bottle, conduct the adaptive evolution in MMC. Adaptive evolution involves continuous sub-cultivation, which can be automatically operated through droplet splitting and fusion. After the adaptive evolution, export the data for analysis. Target droplets can be extracted and then spread on the plate to obtain single colonies. Please click here to view a larger version of this figure.

Figure 2: Structure and essential tools of MMC. (A) External and operation chamber of MMC. (B) The microfluidic chip of MMC. The chip has seven channels (C1-C6 and CF). (C) Reagent bottle. It has a top tube and a side tube. Before injecting the sample into the reagent bottle, it needs to connect a syringe needle to a quick connector A first and then connect the quick connector A to the side tube. (D) Installation of the microfluidic chip. The microfluidic chip is installed on the pedestal. Then the seven channels (C1-C6 and CF) are respectively connected to the corresponding ports of MMC (O1-O6, and OF).

1 – Operation chamber of MMC.
2 – Oil bottle containing the MMC oil.
3 – Waste bottle for collecting waste liquid.
4 – UV lamp (wavelength 254 nm) for sterilization. This lamp can be turned on in advance to sterilize the chip and tubes.
5 – Laser (620 nm) for droplet recognition. The point where the laser is irradiated on the chip is the droplet recognition site.
6 – Temperature probe to measure the temperature inside the operation chamber.
7 – Heater for the operation chamber. It can be used to maintain the temperature of microbial cultivation. The range of temperature that can be set is 25 ± 0.5 °C to 40 ± 0.5 °C.
8 – Optical fiber probe to measure the OD or fluorescence of droplets.
9 – Chip pedestal to install the Microfluidic chip.
10 – Metal bath to fix the reagent bottles and heat them to quickly raise the temperature of a reagent to the temperature of microbial cultivation.
11 – Ports for the microfluidic chip (O1-O6, and OF). The microfluidic chip is connected to the MMC through these ports.
12 – Tubes for droplet storage and cultivation.
13 – Magnet blocks to quickly locate the microfluidic chip during installation.
14 – Syringe needle to inject the samples into the reagent bottles. Its inner diameter is 0.41 mm, and its outer diameter is 0.71 mm.
15 – Quick connector A. Connect with quick connector B.
16 – Quick connector B. Please click here to view a larger version of this figure.

Figure 3: Operationsoftware interface of MMC. (A) The main interface of the software. (1) Temperature in the operation chamber. (2) Photoelectric signal value of droplet recognition. When the droplet passes, the signal value is high (>2 V). When the oil passes, the signal value is low (<1 V). (3) Function selection. There are four functions to choose from: growth curve measurement (Growth Curve), adaptive laboratory evolution (ALE), single factor multi-level analysis (One-factor) and customizing the operations according to experimental needs (Customization). (4) Parameter setting interface. Set the corresponding experimental parameters here after choosing one function. (5) Command run area. (6) Switch of camera. The camera is installed directly above the chip, which can be used to online observe the droplets in the chip. (7) Process display area. Shows the running time, monitoring data, and the operation being executed. (B) The parameter setting interface of adaptive evolution. (C) The droplet screening interface. The MMC can automatically number the droplets. Here the target droplets can be selected and extracted from the MMC. (D) Camera observation interface. Please click here to view a larger version of this figure.

Figure 4: Sample injection, droplet generation, and droplet extraction. (A) The reagent bottle after the injection of bacteria solution and MMC oil. Both the bacteria solution and MMC oil are injected from the side tube. The oil phase is in the upper layer and the bacteria solution is in the lower layer. After the injection, connect the quick connector A and B, and then install it into the MMC. (B) Droplet generation in the microfluidic chip. To enhance the visibility of droplets, a red pigment solution was used to demonstrate the process of droplet generation. (C) Droplet stored in the tube observed by microscope. Scale bar: 400 µm. (D) Pop-up window prompts and the corresponding operations. When the prompt "Please pull out the CF quick connector and put it into the EP tube" appears, pull out the CF connector and put it into the EP tube to collect the target droplet; when the prompt " Please insert the connector back" appears, the droplet collection is complete, insert the CF connector back into the OF port. Please click here to view a larger version of this figure.

Figure 5: Data export and figure plotting of growth curve. (A) Screenshot of part of the exported data. The exported data include each detection time point of the 15 generated droplets and the corresponding OD₆₀₀ values. (B) Growth curve of E. coli MG1655 plotted based on the exported data. Calculate the average OD₆₀₀ values and standard deviation (SD) of 15 droplets at each time point and plot the growth curve. It is clear to see that this growth curve includes the lag phase, logarithmic phase, and stationary phase. Please click here to view a larger version of this figure.

Figure 6: Results of the adaptive evolution of MeSV2.2 in MMC. (A) Growth curves of 50 droplets in the whole adaptive evolution process. The OD₆₀₀ detection data of 50 droplets during the 18-day adaptive evolution process were exported from the MMC and plotted. On the 18th day, 8 droplets, including droplet 6 were extracted. (B) Growth curve of the droplet 6 in the whole adaptive evolution process. The maximum OD₆₀₀ values in the first generation and last sub-cultivation period were 0.37 and 0.58, respectively, increased by 56.8%. (C) Comparison of droplet 6 strain and the initial strain in the shake flask. The strain of droplet 6 and the initial strain were cultivated in shake flasks, and the growth curves (including SD, n = 3) were measured. This figure has been modified from Jian X. J. et al.¹³. Please click here to view a larger version of this figure.

Components	Concentration
Na2HPO4·12H2O	6.78 g/L
KH2PO4	3 g/L
NaCl	0.5 g/L
NH4Cl	1 g/L
vitamin B1 (sterilized by filtration)	0.34 g/L
MgSO4·7H2O	0.049 g/L
CaCl2·2H2O	1.5 mg/L
Microelements:
FeCl3·6H2O	0.5 mg/L
ZnSO4·7H2O	0.09 mg/L
CuSO4·5H2O	0.088 mg/L
MnCl2	0.045 mg/L
CoCl2·6H2O	0.09 mg/L
gluconate	1.09 g/L
methanol	500 mmol/L
isopropyl-β-d-thiogalactopyranoside	0.1 mmol/L
streptomycin sulfate	20 μg/mL
kanamycin sulfate	50 μg/mL
Add extra 15 g/L agarose to prepare solid medium.

Table 1: Components of the special medium for MeSV2.2.

Tunable parameters
Parameter	Range
Temperature of cultivation	25–40 °C ± 0.5 °C
Number of droplets	0–200
Concentration of inoculum	13.3–86.7 %
Concentration of chemical factor	8 different concentrations, up to the maximum concentration of stored chemical factor
The time of sub-cultivation	Up to the user
The number of sub-cultivations	Up to the user
Wavelength of OD detection	350–800 nm
Wavelength of fluorescence detection	Excitation: 470, 528 nm Emission: 350–800 nm
Accuracy parameters
Parameter	C.V
Volume of droplets	1.88%
Concentration of inoculum	<5.0%

Table 2: Tunable parameters and accuracy parameters of MMC. Tunable parameters refer to the parameters that can be adjusted according to the specific requirements of users; accuracy parameters refer to the parameters that reflect the accuracy and reproducibility of the different fluidic operations.

Supplementary Figure 1: Recognition and detection of droplets in MMC. (A) The waveform of a droplet in MMC. This waveform comes from the raw spectral data of the MMC spectrometer. After processing the raw spectral data in the background, MMC will give the measured OD value. (B) OD calculation of droplets in MMC. In the waveform of the droplet, 'a' represents the maximum length of the droplet, 'c' represents the arc-shaped interface formed by oil phase and water phase, and 'b' represents the main part of the droplet. Based on the Lambert-Beer law, the OD value of the droplet is calculated using the following formula: OD value = lg(E/D) × 10. ‘E’ refers to the average spectral signal value of oil phase; ‘D’ refers to the average spectral signal value of the main part b of the droplet. It should be noted that the OD value measured by MMC is different from that measured by a spectrophotometer. Please click here to download this File.

Supplementary Figure 2: Test of crosstalk between the droplets. To verify whether there is crosstalk between the droplets during the long-term cultivation, the E. coli MG1655 solution was diluted to a very low concentration (according to Poisson distribution, λ = 0.1), and then 200 droplets were generated and cultivated for 5 days. After measuring the OD, it was found that the E. coli MG1655 grew in a small number of droplets. And there was almost no bacterial growth in the droplets around these droplets. The result also preliminarily shows that there is little crosstalk between droplets. Please click here to download this File.

Supplementary Table 1: Stability of droplet generation in MMC. As shown in Supplementary Figure 1, the droplet has a fixed waveform. The spectrometer of MMC generates a certain number of data points per second, so the number of data points of the droplet waveform can reflect the size of the droplet. The red dye solution was used to generate 397 droplets in the MMC, and the OD value was measured. The raw spectral data was exported, the data points of each droplet waveform were counted, and the coefficient of variation (C.V) of the droplet data points was calculated. Please click here to download this Table.

Supplementary Table 2: Droplet evaporation in MMC. Here the red dye solution was used to generate droplets in the MMC and the droplets were stored in the cultivation tube. The tube was then placed in a 37 °C constant temperature incubator for 30 days, and the droplet length was regularly measured (take photos under a microscope and measure the length with a scale bar). It shows that the volume of the droplet was reduced by about 12.3% after 30 days, which indicates that the evaporation of the droplet is very small in MMC. Please click here to download this Table.

Discussion

This protocol presents how to use the Microbial Microdroplet Culture system (MMC) to perform automated microbial cultivation and long-term adaptive evolution. MMC is a miniaturized, automated, and high-throughput microbial cultivation system. Compared with conventional microbial high-throughput cultivation methods and instruments, MMC has many advantages such as low labor and reagent consumption, simple operation, online detection (OD and fluorescence), high-time-resolution data collection, and superior parallelization. MMC also has some special advantages different from the conventional droplet microfluidic technology, which usually uses the pL and nL droplets. Most previously reported systems that used pL and nL droplets have poor cultivation performance and few detectable parameters (usually only fluorescence)^18,19,20,21. Although there have been some platforms that can achieve better cultivation performance and multiple parameter detection, it is difficult and requires a lot of effort. For example, some researchers reported the OD detection of pL droplets. It is based on image recognition, which has not only false positives but also needs further verification of accuracy²². However, MMC can accomplish these in a relatively simple manner. MMC uses microliter (µL) droplets that are rarely reported. The superior microbial cultivation performance of MMC has been verified, and it can also directly detect OD and fluorescence. Due to the large volume of the µL droplets, the droplet generation is less susceptible to interference, which has higher stability. Meanwhile, more diverse operations can be performed in the microliter droplets, conducive to the realization of automated operations. Furthermore, because the droplets are enclosure spaces, the volatility of the contents can be suppressed (Supplementary Table 2), conducive to performing the long-term microbial cultivation and adaptive evolution when volatile substances exist in the medium¹⁴. This is difficult to achieve in shake flasks and microplates.

However, certain critical points in the protocol are worth emphasizing. Firstly, it should be noted that the OD value measured by MMC is different from that of a spectrophotometer because their optical path lengths of OD measurement are different (1 mm and 10 mm, respectively). Therefore, when comparing the OD value of MMC with that of shake flask, it is necessary to measure the calibration curve¹³. Fortunately, the adaptive evolution process does not require calibration curves because we focus on the relative trends among the growth curves. Next, certain microorganisms are uncultivated in MMC. The droplets rely on the surface tension of the oil-water interface to maintain stability²³. If the microorganisms produce certain substances that disrupt the surface tension of the oil-water interface, such as some Bacillus subtilis strains producing surfactants²⁴, the droplets cannot maintain stability. Furthermore, if the medium itself is an obstacle to the generation of droplets, it is not viable to be used in MMC, for example, very viscous medium or medium containing large particles. At present, the species we have successfully cultivated in MMC includes E. coli, Lactobacillus plantarum, Corynebacterium glutamicum, yeasts, Methylobacterium extorquens, Aspergillus oryzae, microalgae and so on. It is recommended to cultivate the strain in MMC for preliminary experiment. Finally, the connectors and ports between the chip, the reagent bottle, and the MMC must be connected in strict accordance with the protocol. Otherwise, the bacteria solution may flow into the MMC and contaminate the interior. Additionally, it must be pointed out that the current throughput of MMC is limited (0-200), due to the time taken for the operations of sub-cultivation. In the future, we will optimize the control software and the size of the chip to shorten the time and improve the throughput. Since MMC is a modular system, only related parts or software need to be replaced without the requirement of new equipment.

At present, MMC can not only conduct growth curve measurement, adaptive laboratory evolution, and single-factor multi-level analysis but also be used to customize different droplet operation procedures to meet experimental needs. In the future, it is necessary to further enrich the application functions of the MMC system in response to the different needs of microbial research, such as conducting the multi-factor multi-level orthogonal experiments, multi-sample automatic sampling technology to simultaneously measure the growth curves of multiple bacterial species, and accurately detect and control more parameters (e.g., dissolved oxygen (DO) and pH). At the same time, it is also necessary to develop more functions in the field of microbiology to apply MMC to more practical scenarios, such as optimization of medium compositions, determination of minimum inhibitory concentration (MIC), co-cultivation of microorganisms²⁵, etc.

Materials

0.22 μm PVDF filter membrane	Merck Millipore Ltd.	SLGPR33RB	Sterilize the MMC oil
4 °C refrigerator	Haier	BCD-289BSW	For reagent storage
Agar	Becton, Dickinson and Company	214010	For solid plate preparation
CaCl2·2H2O	Sinopharm Chemical Reagent Beijing Co., Ltd.	20011160	Component of the special medium for MeSV2.2.
Clean bench	Beijing Donglian Har Instrument Manufacture Co., Ltd.	DL-CJ-INDII	For aseptic operation and UV sterilization
CoCl2·6H2O	Sinopharm Chemical Reagent Beijing Co., Ltd.	10007216	Component of the special medium for MeSV2.2.
Computer	Lenovo	E450	Software installation and MMC control
Constant temperature incubator	Shanghai qixin scientific instrument co., LTD	LRH 250	For the microbial cultivation using solid medium
CuSO4·5H2O	Sinopharm Chemical Reagent Beijing Co., Ltd.	10008218	Component of the special medium for MeSV2.2.
Electronic balance	OHAUS	AR 3130	For reagent weighing
EP tube	Thermo Fisher	1.5 mL	For droplet collection
FeCl3·6H2O	Sinopharm Chemical Reagent Beijing Co., Ltd.	10011928	Component of the special medium for MeSV2.2.
Freezing Tube	Thermo Fisher	2.0 mL	For strain preservation
Gluconate	Sigma-Aldrich	S2054	Component of the special medium for MeSV2.2.
Glycerol	GENERAL-REAGENT	G66258A	For strain preservation
High-Pressure Steam Sterilization Pot	SANYO Electric	MLS3020	For autoclaved sterilization
isopropyl-β-d-thiogalactopyranoside (IPTG)	Biotopped	420322	Component of the special medium for MeSV2.2.
Kanamycin sulfate	Solarbio	K8020	Component of the special medium for MeSV2.2.
KH2PO4	MACKLIN	P815661	Component of the special medium for MeSV2.2.
Methanol	MACKLIN	M813895	Component of the special medium for MeSV2.2.
MgSO4·7H2O	BIOBYING	1305715	Component of the special medium for MeSV2.2.
Microbial Microdroplet Culture System (MMC)	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-I	Performing growth curve determination and adaptive evolution. Please refer to http://www.tmaxtree.com/en/index.php?v=news&id=110
Microfluidic chip	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-ALE-OD	For various droplet operations. Please refer to http://www.tmaxtree.com/en/
MMC oil	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-M/S-OD	The oil phase for droplet microfluidics. Please refer to http://www.tmaxtree.com/en/
MnCl2	Sinopharm Chemical Reagent Beijing Co., Ltd.	20026118	Component of the special medium for MeSV2.2.
NaCl	GENERAL-REAGENT	G81793J	Component of the LB medium
Na2HPO4·12H2O	GENERAL-REAGENT	G10267B	Component of the special medium for MeSV2.2.
NH4Cl	Sinopharm Chemical Reagent Beijing Co., Ltd.	10001518	Component of the special medium for MeSV2.2.
Petri dish	Corning Incorporated	90 mm	For the preparation of solid medium
Pipette	eppendorf	2.5 μL, 10 μL, 100μL, 1000μL	For liquid handling
Quick connector A	Luoyang TMAXTREE Biotechnology Co., Ltd.	—	For the connection of each joint. Please refer to http://www.tmaxtree.com/en/
Reagent bottle	Luoyang TMAXTREE Biotechnology Co., Ltd.	MMC-PCB	Sampling and storage of bacteria solution and reagents. Please refer to http://www.tmaxtree.com/en/
Shake flask	Union-Biotech	50 mL	For microbial cultivation
Shaking incubator	Shanghai Sukun Industrial Co., Ltd.	SKY-210 2B	For the microbial cultivation in shake flask
Streptomycin sulfate	Solarbio	S8290	Component of the special medium for MeSV2.2.
Syringe	JIANGSU ZHIYU MEDICAL INSTRUCTMENT CO., LTD	10 mL	Draw liquid and inject it into the reagent bottle
Syringe needle	OUBEL Hardware Store	22G	Inner diameter is 0.41 mm and outer diameter is 0.71 mm.
Tryptone	Oxoid Ltd.	LP0042	Component of the LB medium
Ultra low temperature refrigerator	SANYO Ultra-low	MDF-U4086S	For strain preservation (-80 °C)
UV–Vis spectrophotometer	General Electric Company	Ultrospec 3100 pro	For the measurement of OD values
Vitamin B1	Solarbio	SV8080	Component of the special medium for MeSV2.2.
Yeast extract	Oxoid Ltd.	LP0021	Component of the LB medium
ZnSO4·7H2O	Sinopharm Chemical Reagent Beijing Co., Ltd.	10024018	Component of the special medium for MeSV2.2.