Polar region microorganism isolated culture method based on droplet microfluidic technology

Abstract

The invention discloses a polar region microorganism isolated culture method based on a droplet microfluidic technology. The method comprises the following steps: acquiring a polar region sample, and oscillating and extracting at low temperature to obtain a sample suspension; determining the plate culturable rate R through a solid plate culture method; based on the R and a preset target number N of growable cells, calculating a target total number T of cells required by the droplet microfluidic experiment, and preparing an on-machine sample with corresponding cell concentration; the method comprises the following steps: generating a single-cell-wrapped micro-droplet from an on-machine sample through a droplet microfluidic system, and performing long-term culture at low temperature; after culturing, screening positive liquid drops and purely culturing the strain. The micro-fluidic sample loading concentration is accurately optimized through culturable rate feedback, the separation efficiency of microorganisms difficult to culture in the polar region is remarkably improved, interspecific competitive inhibition of a traditional plate is broken through, and the diversity of obtained strains and the proportion of novel groups are greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial culture, in particular to a microbial separation and culture method based on microfluidic technology, and more particularly to an optimization method for improving the separation efficiency of polar difficult-to-culture environmental microorganisms, species diversity and acquisition rate of novel groups. BACKGROUND

[0002] There are rich microbial species and high species diversity in nature, but the proportion of microorganisms that have been isolated and cultured is less than 1%, which is an important factor limiting the development of microbial resources and basic research. The microbial separation and culture technology based on culture dishes developed more than a hundred years ago is still used today and is still the main method, and even the only method used by researchers in many cases. However, there are many challenges and limitations in using the traditional plate method to separate and culture microorganisms from complex environments: the plate method can only isolate a limited number of species, and most species cannot grow under experimental conditions; the plate method requires repeated selection and multiple purification from mixed bacterial populations to obtain single pure culture, which is often time-consuming, labor-intensive and material-consuming; the plate method is prone to form dominant colonies, and fast-growing dominant groups can compete for nutrients from other slow-growing groups, or produce products that inhibit the growth of other groups, resulting in limited diversity on the plate, and often representing fast-growing groups under experimental conditions. The method is not effective for rare microbial groups and difficult-to-culture microbial groups. Therefore, it is urgent to develop new methods to improve separation and culture efficiency, eliminate dominant group inhibition, and reduce repeated groups.

[0003] Under the selection pressure of long-term low temperature freezing, oligotrophic, drought, high radiation, and extreme light cycle in the north and south poles, polar microorganisms have higher novelty and uniqueness at the species and gene levels, and therefore become important objects for studying extreme life characteristics, unique species resources, gene resources, and product resources. However, due to unique environmental selection and evolutionary history, most polar microorganisms prefer low temperature and grow slowly, and the separation and culture efficiency on conventional plates is even lower. Therefore, it is more necessary to develop an efficient separation and culture method to improve the separation and culture efficiency of polar microorganisms and obtain groups that are difficult to obtain under conventional conditions.

[0004] Droplet microfluidic technology is characterized by rapid formation of tens of thousands of droplets, and can achieve single cell encapsulation, forming droplets with a volume of picoliters to nanoliters, which can effectively isolate the competition and inhibition between cells in theory, and provide a uniform growth environment for each cell, thereby breaking through the bottleneck of traditional plate culture and capturing more diverse and rare microbial groups. However, the technology has not been optimized and adapted for polar microorganisms, making it difficult to evaluate its actual application effect. Existing microfluidic culture technologies (such as WO2018075701A1, WO2018026594A1, etc.) focus on high-throughput screening, phenotype analysis or condition optimization of known cultivable strains, and the technical design (such as cell concentration range, culture time, screening index) is based on fast-growing model microorganisms or industrial strains. Directly applying such general schemes to extremely slow-growing polar microorganisms will face a series of adaptability problems such as inaccurate cell encapsulation rate calculation and insufficient culture period, and cannot achieve the core goal of efficiently isolating and culturing difficult-to-culture microorganisms.

[0005] Therefore, in view of the particularity of polar difficult-to-culture microorganisms, developing a droplet microfluidic separation and culture method adapted to them to substantially improve their separation and culture efficiency and diversity has become a technical problem to be solved in the field. SUMMARY

[0006] In view of the defects of the prior art, especially the low efficiency, limited diversity and difficulty in obtaining novel groups when using traditional plate method and general microfluidic technology to isolate and culture polar difficult-to-culture microorganisms, the purpose of the present application is to provide an optimized polar microorganism isolation and culture method based on droplet microfluidic technology, which improves the isolation and culture efficiency of polar microorganisms and obtains new species that are difficult to obtain under normal circumstances.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: A polar microorganism isolation and culture method based on droplet microfluidic technology, comprising the following steps: S1. Sample pretreatment: obtaining a polar environment sample, and performing shock extraction at 2-8°C to obtain a raw sample suspension containing microbial cells; S2. Culturability determination: gradient dilution of the raw sample suspension obtained in step S1, and plate culture at 4-15°C for 10-30 days by using solid plate culture method, and calculating the plate culturability R of the polar microorganism under the selected culture conditions by colony counting; S3. Microfluidic sample preparation: based on the plate cultivability R measured in step S2, and the target number of growable cells N of the droplet microfluidic device, the target total number of cells T required for the droplet microfluidic experiment is calculated, wherein T = N / R; according to the target total number of cells T, the original sample suspension obtained in step S1 is processed to prepare a sample for machine loading with a total cell concentration C, wherein the range of C is set to 1-10 times of T / V, V is the set loading volume, and the sample for machine loading contains a liquid culture medium; S4. Droplet microfluidic culture: the sample for machine loading prepared in step S3 is loaded into the droplet microfluidic device to generate microdroplets and achieve single cell encapsulation, and the culture is carried out at 10-15 DEG C for 15-30 days.

[0008] Further, in step S1, the polar environment sample is a polar soil or sediment sample; the time of the shock extraction is 1-3 hours.

[0009] Further, in step S2, the plate cultivability R is calculated by the following formula: R = (plate colony number) / (coating volume x bacterial liquid concentration).

[0010] Further, in step S3, the initial total number of cells T required is calculated = N / R, and the sample for machine loading with the corresponding cell concentration is prepared according to the initial total number of cells T.

[0011] Further, in step S3, the specific method for preparing the sample for machine loading is: resuspending the sample cells containing the initial total number of cells T in 50-150 mL of liquid culture medium.

[0012] Further, in step S3, the target number of growable cells N is 3000-7000.

[0013] Further, step S3 further comprises setting a basic concentration C and / or a high concentration of the sample for machine loading which is 5-15 times of the basic concentration C. In step S4, different concentrations of the sample for machine loading are set with different culture times, wherein the culture time of the high concentration sample is shorter than that of the sample with the basic concentration. The culture time of the high concentration sample is set to 15-20 days, and the culture time of the sample with the basic concentration is set to 30 days.

[0014] Further, the polar microbial isolation and culture method based on the droplet microfluidic technology further comprises step S5, pure culture strain acquisition: after the culture in step S4 is completed, the microdroplets with positive microbial growth are screened and sorted into a multi-well plate for large-scale culture, thereby obtaining a pure culture strain.

[0015] The beneficial effects of the present application are: The present application solves the adaptability problem of the prior art by precise quantitative calibration. It innovatively uses the "culturable rate" obtained by traditional plate culture as a key feedback parameter to accurately calculate and optimize the microfluidic loading concentration in reverse, fundamentally overcoming the problem of inaccurate calculation of cell encapsulation rate when applying a general microfluidic scheme to unknown difficult-to-culture microorganisms. At the same time, in view of the extremely slow growth characteristics of polar microorganisms, the system designs long-term low-temperature culture combined with "concentration-time" double gradient optimization strategy to ensure the sufficient growth and capture of slow-growing and rare groups. The present application combines high-throughput droplet microfluidic technology with the characteristics of polar microorganisms, and in the two stages of single cell separation and pure culture acquisition, compared with the traditional plate method, the time efficiency is significantly improved, the reagent consumption such as culture medium is significantly reduced, and the manpower and material resources cost is greatly saved.

[0016] The present application method has made a revolutionary breakthrough in species acquisition ability and diversity. The single cell isolation effect of droplets effectively eliminates the competitive inhibition of dominant flora, enabling a large number of slow-growing and rare microbial groups to grow. Experiments have shown that the number of microorganisms obtained by the present application method at each classification level such as genus, family, order, class and phylum is significantly more than that obtained by the traditional plate method, and the community structure is more uniform.

[0017] The present application method can enrich and separate novel groups that cannot be obtained or have very low abundance by traditional plate method, including multiple potential new high-order taxonomic units (such as new class, new order) and rare bacterial genera with important theoretical research and application development value (such as Bacillus, Caulobacter, Rhodoferax, Massilia, Arenimonas, etc.), providing an unprecedented material basis for the expansion and subsequent development and utilization of polar microbial resources.

[0018] In summary, the present application optimizes the microfluidic loading concentration through the culturable rate feedback, significantly improves the separation efficiency of polar difficult-to-culture microorganisms, breaks through the inter-species competition inhibition of traditional plate, greatly improves the diversity and novel group proportion of obtained strains, and provides an efficient and high-throughput method for polar microbial resource mining. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Distribution of strains at genus level obtained by different methods.

[0020] Figure 2 Heat map of strain groups with abundance higher than 0.1%. DETAILED DESCRIPTION

[0021] Example 1 1. Polar environment sample pretreatment method Aseptically collect polar soil samples and store them at 4°C. In a sterile environment, take 1 gram of soil and place it in 5-10 ml of sterile physiological saline. Shake the sample for 1-2 hours at 4°C to thoroughly extract microbial cells. After shaking, let the sample stand at 4°C for 30-60 minutes. Transfer the supernatant (i.e., the stock solution) to a new sterile centrifuge tube, and aspirate 2-5 μL for microbial cell counting under a microscope.

[0022] 2. Optimization of traditional plate culture pre-experiment The above-mentioned stock solution was serially diluted (1:10, 1:100, 1:1000). Ten microliters of both the stock solution and the serially diluted samples were then plated on conventional R2A agar plates and incubated at 15°C for 10, 15, 20, and 30 days. Colony counts were performed to calculate the culturability. Sample concentration gradients with colony counts above 100 were selected for calculation. The culturability (R) was calculated using the formula: Plate culturability (R) = Plate colony count / (Spread volume × Bacterial concentration). Based on the culturability and time obtained from the above preliminary experiments, the optimal conditions for subsequent high-throughput isolation and culture were determined. In this example, the culturability (R) of the polar soil sample cultured on R2A medium at 15°C for 30 days was approximately 0.089%.

[0023] 3. Optimization of droplet microfluidic schemes Based on a culturability of 0.089%, the required sample concentration for high-throughput droplet culture can be deduced: For droplet microfluidic devices (taking Tianmu Bio's MISS cell instrument as an example), the recommended cell concentration range for samples is 50-100 cells / mL. This example uses a minimum of 50 cells / mL. Based on a culturability of 5000 cells, and assuming a culturability of 0.089%, the required number of cells for sample preparation is 5000 / 0.089% ≈ 5.6 × 10⁻⁶. 6 indivual.

[0024] Because polar microorganisms require low-temperature cultivation and grow slowly, the droplet microfluidic separation and culture protocol was optimized for both time and concentration gradients. Based on the calculated cell requirements, two cell concentrations (i.e., the previously mentioned 5.6 × 10⁻⁶) were prepared. 6 The concentration of the cells was designated as low concentration; then a sample with a concentration 10 times higher was added and designated as high concentration. The high concentration sample was collected after being cultured at 15°C for 17 days, and the low concentration sample was collected after being cultured at 15°C for 30 days.

[0025] Following the two optimization schemes described above, a sample of the corresponding volume (100 ml in this embodiment) was taken as the sample for loading. After the sample and oil droplets were injected separately and then encapsulated, the encapsulation rate, single-droplet rate, and multi-droplet rate were calculated based on the Poisson distribution principle. The droplet microfluidic device can generate 20,000 droplets within 4 hours, greatly improving the separation efficiency.

[0026] 4. Traditional plate isolation culture control To compare with droplet microfluidics, using the same injected sample, an equal volume of bacterial suspension was aspirated and plate-spread simultaneously under the same conditions: the bacterial suspension was diluted 1:10 and evenly spread onto four R2A plates. Similarly, the high-concentration sample was incubated at 15°C for 17 days, and the low-concentration sample for 30 days. Colony counts were then calculated.

[0027] 5. Methods for detecting the composition and diversity of isolated and cultured bacterial strains Two in-situ culture schemes were employed, involving in-situ culture in droplets and plate culture for 17 and 30 days, respectively, after which culture was stopped. The bacterial strains growing in the droplets were analyzed for OD using Misscell software. Droplets with significantly higher OD values ​​than the background were selected as growth droplets. Each growing droplet was automatically collected and inoculated into a 96-well plate containing 100 μL of R2A culture medium. A total of 1300 droplets were collected after 30 days of low-concentration culture, and 2939 droplets were collected after 17 days of high-concentration culture, for a total of 4239 droplets.

[0028] In the concurrent traditional plate culture, fungal contamination was evident in the later stages of 30-day low-concentration culture, with approximately 80-100 bacterial colonies per plate, totaling about 300-400 colonies. In the high-concentration culture after 17 days, there were over 100 colonies per plate, with a total of approximately 500 colonies.

[0029] To quickly compare the bacterial diversity and species composition obtained by the two methods, 10 μL of each droplet from the droplet method was collected and combined into a final mixed sample, resulting in two samples for testing: DropAS (high concentration cultured for 17 days) and DropAS_2 (low concentration cultured for 30 days). All colonies obtained from the plate method were washed with sterile saline, and the colonies on the plates were collected and finally combined into mixed samples: PlateAS (high concentration cultured for 17 days) and PlateAS_2 (low concentration cultured for 30 days).

[0030] The above four samples underwent high-throughput sequencing of 16S rRNA gene amplicon. In order to more accurately determine the species composition and obtain higher species classification resolution, the full-length 16S rRNA gene was amplified using universal primers 27F and 1492R, and the PacBio long fragment sequencing platform was selected for sequencing.

[0031] 6. Comparison of the efficiency of cultivation methods The time cost and reagent input of the two methods were compared based on the input of 1 person for separating and culturing more than 4200 strains. The time cost of the traditional plate method was significantly higher than that of the high-throughput droplet microfluidic technology because the traditional plate method required a large number of plate preparation and coating, multiple plate separation and purification, and the like. Specifically, in the single cell separation stage, the high-throughput droplet method can generate 20,000 droplets in 4 hours, only consuming 100 ml of culture solution. To obtain the same number of single colonies coated on the plate, at least 100-200 plates (possibly more) are required. Based on the normal working capacity of one person, at least 3 days are required from plate preparation to coating completion, and 5 L of culture medium is consumed. Compared with the traditional plate separation and culture method, the application of the high-throughput droplet microfluidic technology can improve the time efficiency by 18 times and reduce the culture medium reagent input by 50 times.

[0032] In the single cell screening stage after the completion of the culture, pure culture strains need to be obtained. The traditional plate method often requires multiple streaking purification from the mixed bacteria on the plate to obtain 4200 pure culture strains. Based on the normal working capacity of one person for 8 hours per day, it is conservatively estimated that at least one month is required to complete the work, and a large number of plates need to be prepared (4200 plates are required for the case of successful purification once). Under the same conditions, the droplet high-throughput technology only needs to automatically screen the droplets that grow and automatically input the 96-well plate to obtain the pure culture strains, and the time is shortened to 8 hours. Therefore, the time efficiency is improved by at least 90 times, and the culture medium input is reduced by at least 150 times.

[0033] Table 1. Efficiency comparison of two separation and culture technologies.

[0034] * The input of 1 person for separating and culturing more than 4200 strains was calculated.

[0035] 7. Comparison of culture method effects and application value By mixing and sequencing all the strains obtained by the two methods, the strain diversity and novelty of the strains separated and cultured by the two methods can be compared and analyzed to evaluate the culture effect. Based on the full-length amplicon high-throughput sequencing of 16S rRNA gene, more than 70,000 full-length sequences are obtained for each sample. After the Qiime2 analysis process, short fragment sequences, adapter sequences, chimeric sequences, and repeat sequences are removed, and finally reads meeting the quality requirements are obtained for subsequent analysis. After annotation by a self-trained annotator, species classification information is obtained.

[0036] The results show (Table 2) that the number of groups obtained by droplet microfluidic technology is much higher than that of traditional plates. Under high concentration, microfluidic technology obtains 45 more genera, 15 more families, 10 more orders, 6 more classes, and 3 more phyla than traditional plates. Under low concentration, microfluidic technology obtains 59 more genera, 15 more families, 8 more orders, 3 more classes, and 2 more phyla than traditional plates. Moreover, droplet microfluidic technology significantly improves the evenness of the obtained species and reduces the significant inhibition of dominant strains Figure 1 ), which is specifically manifested in that the dominant strain Pseudomonas genus is reduced by 18% under high concentration culture by microfluidic technology (49%) compared to traditional plate method (67%), and is reduced by 38% under low concentration culture by microfluidic technology (14%) compared to traditional plate method (52%). At the same time, the proportion of other non-dominant groups increases significantly, and the evenness increases significantly. This shows that after the cells are individually encapsulated, the microfluidic technology eliminates the inhibition of other strains caused by the rapid growth of dominant strains, can obtain more strain types under the condition of screening the same number of strains, and improves the diversity of obtained strain groups.

[0037] In addition, microfluidic technology also improves the ability to obtain novel groups. For example Figure 2As shown, the species abundance at the level of new door, new class, and new order obtained by microfluidic technology was significantly higher than that by plate method, which means that the application of new technology can increase the isolation of novel groups at higher taxonomic levels that have not been isolated and cultured before, which is of great significance for the research and application of polar microbial resources, the acquisition of new genetic resources, and the development of new biotechnology. At the same time, microfluidic technology can also enrich some groups with very low abundance or non-existent in conventional plates. For example, Bacillus accounted for about 3.7% in microfluidic technology, but 0 in plate method; Caulobacter accounted for 1.1% and 3.6% in microfluidic technology, but 0 in plate method; Rhodoferax accounted for 0.84% (DropAS) and 4.1% (DropAS_2) in microfluidic technology, and less than 0.1% in plate method; Massilia, Arenimonas, Brevundimonas, and many unidentified groups (such as bacteria domain, Proteobacteria, Bacteroidetes, Gamma Proteobacteria, and multiple families (Comamonadaceae, Oxalobacteraceae, Sphingobacteriaceae)) were also significantly enriched in microfluidic technology. These strain resources have very important research significance and application value: for example, Bacillus is an important component of many agricultural growth-promoting bacteria and an important chassis cell in synthetic biology; Caulobacter is a model organism for studying cell differentiation and asymmetric division; Rhodoferax is an excellent candidate for microbial fuel cells and has application value in enzyme resources; Massilia can synthesize biological plastics such as β-hydroxybutyric acid (PHB), and some can degrade chitin and organic pollutants, inhibit plant pathogenic bacteria, etc.; Arenimonas can degrade polycyclic aromatic hydrocarbons (PAHs), diesel, and other petroleum hydrocarbons, and has application value in oil-contaminated soil ecological restoration, and some species can synthesize biological plastics such as poly-β-hydroxybutyric acid (PHB), produce chitinase, esterase, acid phosphatase, etc.

[0038] In addition, the application optimizes the microfluidic technology for different cell concentrations and culture times, and finds that low concentration and long-term culture can have higher diversity than short-term high concentration culture, and can improve the probability of obtaining novel groups. For example, under long-term low concentration, the microfluidic technology significantly improves the proportion of groups such as Devosia, Cellulomonas, [Aquaspirillum]_arcticum_group, Mycobacterium, Nocardioides, Mesorhizobium, Sphingomonas, Galbitalea, Rhizobacter, Rhizorhapis, Caenimonas, Paucibacter, Dyadobacter, etc. Figure 2 These groups can secrete cellulase, hemicellulase, chitinase and other enzymes at low temperature, and are models for studying the low-temperature adaptation mechanism of enzymes (such as flexible active center); in cold ecosystems, they are the key starters for decomposing plant residues and organic detritus, and determine the carbon mineralization rate and greenhouse gas flux; in the application aspect, they can be used for organic pollution remediation (such as petroleum hydrocarbon, pesticide) in low-temperature environment, plant growth or soil improvement agent in barren and cold soil, etc.

[0039] Table 2. Comparison of the number of species groups obtained by each method.

[0040]

Claims

1. A method for isolating and culturing polar microorganisms based on droplet microfluidic technology, characterized in that, Includes the following steps: S1, Sample pretreatment: Obtain polar environment samples and perform shaking extraction at 2-8℃ to obtain the original sample suspension containing microbial cells; S2, Culturability determination: The original sample suspension obtained in step S1 is serially diluted and cultured at 4-15℃ for 10-30 days using the solid plate culture method. The plate culturability R of the polar microorganism under the selected culture conditions is calculated by colony counting. S3, Microfluidic Sample Preparation: Based on the plate culturability R measured in step S2, and combined with the target number of culturing cells N of the droplet microfluidic device, calculate the target total number of cells T required for the droplet microfluidic experiment, where T = N / R; according to the target total number of cells T, process the original sample suspension obtained in step S1 to prepare a sample with a total cell concentration of C, where C is set to be 1-10 times T / V, and V is the set loading volume, and the sample contains liquid culture medium; S4, Droplet microfluidic culture: The sample prepared in step S3 is loaded onto the droplet microfluidic device to generate microdroplets and achieve single-cell encapsulation, and cultured at 10-15℃ for 15-30 days.

2. The method according to claim 1, characterized in that, In step S1, the polar environment sample is a polar soil or sediment sample; the shaking extraction time is 1-3 hours.

3. The method according to claim 1, characterized in that, In step S2, the culturability R of the plate is calculated by the following formula: R = (number of colonies on the plate) / (coating volume × bacterial concentration).

4. The method according to claim 1, characterized in that, In step S3, the required initial total number of cells T = N / R is calculated, and the sample with the corresponding cell concentration is prepared according to the initial total number of cells T.

5. The method according to claim 1, characterized in that, In step S3, the specific method for preparing the sample for use is as follows: resuspend the sample cells containing the initial total cell number T in 50-150 mL of liquid culture medium.

6. The method according to claim 1, characterized in that, In step S3, the target number of gradable cells N is 3000-7000.

7. The method according to claim 1, characterized in that, Step S3 also includes setting a base concentration C and / or a high concentration of 5-15 times the base concentration C for the sample to be used.

8. The method according to claim 7, characterized in that, In step S4, different incubation times are set for samples of different concentrations, with the incubation time for high-concentration samples being shorter than that for samples of basic concentration.

9. The method according to claim 1, characterized in that, The method further includes step S5, obtaining pure culture strains: after the culture in step S4, microdroplets with positive microbial growth are screened and sorted into multi-well plates for expansion culture, thereby obtaining pure culture strains.

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