Method for screening chlorella pyrenoidosa mutants
By using droplet microfluidics and fluorescence signal detection, the problems of low throughput, low efficiency and poor accuracy of traditional algae screening methods have been solved, and efficient screening of high-growth-performance Chlorella proteoglycan mutants has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-02
- Publication Date
- 2026-04-07
AI Technical Summary
Traditional algae screening methods suffer from low throughput, low efficiency, poor accuracy, high cost, and long processing time, making it difficult to achieve high-throughput and single-cell precision screening, and they cannot dynamically track the growth process.
Droplet microfluidic technology was used to encapsulate Chlorella cells in microdroplets, and combined with fluorescence signal detection, to achieve single-cell growth phenotype analysis. Microdroplet culture was controlled by light and gas pressure, and the growth phenotype of algal cells was monitored in real time.
This method enables high-throughput, low-cost, and label-free single-cell screening, and can quickly screen for high-growth-performance Chlorella proteoglycan mutants, solving the problems of low throughput and poor accuracy of traditional methods.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of microbial engineering, and particularly relates to a method for screening Chlorella pyrenoidosa mutants. BACKGROUND
[0002] Algae are a group of organisms that mainly rely on photosynthesis for energy, and have the characteristics of rich species diversity, efficient light energy conversion efficiency and rapid growth and reproduction. With the promotion of global energy transformation, algae have unique advantages in the fields of bioenergy development, high value-added product synthesis, environmental remediation and synthetic biology research, and have become an important research object of green biological manufacturing. However, the industrial production of algae still faces many challenges, among which the growth rate of algae strains and the accumulation efficiency of metabolic products are the main bottlenecks restricting their large-scale application. Therefore, the screening and improvement of high-efficiency algae strains have become a key link to promote the industrialization development of algae. Traditional screening methods mostly use plate culture, shake flask culture or micro-manipulation technology to measure the cell density, biomass or morphological characteristics to evaluate the growth performance. Such methods have low throughput, long time-consuming, and are difficult to achieve efficient screening at the single cell level. Single cell sequencing can achieve accurate genotype-phenotype correlation analysis, but it is costly and time-consuming, and is difficult to meet the demand of large-scale screening. Flow cytometry can improve the screening throughput, but the pretreatment steps are complex, and it is difficult to achieve long-term dynamic monitoring. Therefore, a high-throughput, label-free and dynamic tracking method for single cell growth is needed to quickly screen excellent algae strains.
[0003] Chlorella pyrenoidosa is a single-cell green algae with great application value. It has become an important biological resource in many fields due to its fast growth rate, high photosynthetic efficiency, rich nutrition and easy cultivation. The growth rate and metabolic product yield of natural algae strains often cannot meet the needs of industrialization, and excellent mutants need to be obtained through mutagenesis or gene editing. Traditional algae screening techniques have many challenges and limitations, such as: unable to balance high throughput and single cell precision, resulting in low screening efficiency; spectral technology accuracy is easily affected by environmental factors; single cell sequencing technology is costly and time-consuming, and data analysis is complex; flow cytometry cannot dynamically track the growth process, etc. SUMMARY
[0004] To solve the above technical problems, the application provides a method for high-throughput screening of Chlorella pyrenoidosa mutants with high growth performance.
[0005] The application relates to the following contents: 1. An apparatus, characterized in that the culture apparatus comprises an upper layer and a lower layer, the upper layer has a light source at the top, a coil holder, and air inlets and outlets on both sides of the coil holder.
[0006] 2. The apparatus according to claim 1, characterized in that a fan is mounted on the bottom of the lower layer.
[0007] 3. The apparatus according to item 1, characterized in that the air inlet is connected to a gas cylinder, and the flow rate of the incoming gas is adjusted by a pressure reducing valve connected to the gas cylinder.
[0008] 4. A method for screening protein-nucleated Chlorella mutants, characterized by comprising the following steps: Chlorella pyrenoidosa was prepared by activating, culturing and mutagenesis to obtain Chlorella pyrenoidosa algal solution; The mutagenized algal solution was encapsulated in microdroplets; Under given conditions, the encapsulated microdroplets are cultured for a given time. The fluorescence signal of microdroplets after culture was detected, and different growth rates of Chlorella proteoglycans were screened based on the intensity of the fluorescence signal.
[0009] 5. The method according to item 4, characterized in that the concentration of the mutagenized Chlorella proteoglycan solution is 3.5 × 10⁻⁶. 5 —4×10 5 CFU / mL.
[0010] 6. The method according to item 4, wherein the diameter of the microdroplets is 70-80 μm, preferably 80 μm.
[0011] 7. The method according to item 4, wherein the microdroplets are droplets of 1-999 picoliters.
[0012] 8. The method according to item 4, characterized in that the given conditions include: culturing the encapsulated microdroplets for a given time under certain light conditions and gas pressure. The illumination conditions are 200,000 lux to 800,000 lux; preferably 200,000 lux to 400,000 lux. The gas pressure is 0.01 MPa to 0.04 MPa; preferably 0.01 to 0.02 MPa. The given time is for cultivation until the fluorescence signals generated by different microdroplets can be distinguished by a fluorescence detection device.
[0013] 9. The method according to item 8, wherein the given condition is implemented by any one of items 1 to 3.
[0014] 10. Use of the apparatus described in any one of items 1 to 3 in the culture of microdroplets.
[0015] 11. The use according to item 10, characterized in that the microdroplet is the encapsulated microdroplet as described in any one of items 4 to 7.
[0016] Invention Effects 1. This application combines the high accuracy of droplet single-cell culture with the high-throughput screening advantages of microfluidics, overcoming the shortcomings of traditional methods such as low throughput and high false positives.
[0017] 2. This application utilizes droplet microfluidics to encapsulate individual algal cells within independent microdroplets, enabling true single-cell-level growth phenotypic analysis. This solves the signal interference problem caused by cell aggregation in traditional methods, achieving single-cell precision in screening resolution.
[0018] 3. This application detects chlorophyll fluorescence signals spontaneously generated by algal cells, coupling the growth phenotype of microalgae with light signals. It eliminates the need for exogenous labeling or destructive sampling, thus avoiding the impact on cell viability.
[0019] 4. This application can observe cell morphology and aggregation status in real time during droplet culture, and combine chlorophyll fluorescence signals to achieve multi-dimensional dynamic analysis of growth phenotype. Attached Figure Description
[0020] Figure 1 Flowchart of a method for high-throughput screening of rapid-growing Chlorella mutants.
[0021] Figure 2 This is a schematic diagram showing the encapsulation state of microdroplets of Chlorella proteoglycans under different algal solution concentrations.
[0022] Figure 3 This is a schematic diagram illustrating the effect of microdroplets of different diameters on the encapsulation effect of single cells of Chlorella proteoglycans.
[0023] Figure 4 This is a schematic diagram of a droplet culture device.
[0024] Figure 5 This is a timeline diagram of the growth of Chlorella proteoglycans within a droplet.
[0025] Figure 6 This is a fluorescence spectrum of chlorophyll in Chlorella cells.
[0026] Figure 7 This is a real-time signal image of droplet sorting from Chlorella proteoglycans.
[0027] Figure 8 This is a histogram of the droplet signal from Chlorella proteoglycans.
[0028] Figure 9 Microscopic images of Chlorella droplets before and after sorting.
[0029] Explanation of reference numerals in the attached figures: 1-Upper layer, 11-Light source, 12-Coil rack, 13-Air inlet, 14-Air outlet; 2-Lower layer, 21-Fan. Detailed Implementation
[0030] The present application will now be described in detail with reference to the accompanying drawings, wherein the same numerals in all the drawings denote the same features. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this application will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art.
[0031] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification 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. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
[0032] To address the problems of low throughput, low efficiency, poor accuracy, high cost, and long time consumption in traditional algae screening techniques, this application provides a new method for screening protein-nucleated Chlorella mutants.
[0033] This application first provides an apparatus, wherein the culture apparatus includes an upper layer and a lower layer, the upper layer has a light source at the top, a coil rack, and air inlets and outlets on both sides of the coil rack.
[0034] In this application, a fan is installed at the bottom of the lower layer.
[0035] In this application, the air inlet is connected to a gas cylinder, and the flow rate of the incoming gas is adjusted by a pressure reducing valve connected to the gas cylinder.
[0036] In this application, there are no limitations on the material, shape, etc. of the device, as long as it can transmit light and air.
[0037] This application further provides a method for screening protein-nucleated Chlorella mutants, comprising the following steps: Chlorella pyrenoidosa was prepared by activating, culturing and mutagenesis to obtain Chlorella pyrenoidosa algal solution; The mutagenized algal solution was encapsulated in microdroplets; Under given conditions, the encapsulated microdroplets are cultured for a given time. The fluorescence signal of microdroplets after culture was detected, and different growth rates of Chlorella proteoglycans were screened based on the intensity of the fluorescence signal.
[0038] In this application, no limitation is made on the activation and culture methods of Chlorella proteoglycans, and those skilled in the art can operate according to known methods. For example, the algal solution of Chlorella proteoglycans can be first cultured in shake flasks, and after multiple passages to grow to the logarithmic growth phase, it can be transferred to a new culture medium for large-scale culture to serve as a seed stock solution for subsequent mutagenesis experiments.
[0039] In this application, no limitation is made on the culture medium used in the cultivation of Chlorella proteoglycans. Those skilled in the art can choose from well-known sources, such as liquid culture medium or solid culture medium.
[0040] In some preferred embodiments, liquid culture medium is selected for culturing Chlorella proteoglycans.
[0041] In this application, the components of the liquid culture medium are not limited. Those skilled in the art can select them according to specific experimental conditions, as long as the growth and reproduction of Chlorella proteoglycans can be achieved.
[0042] In some preferred embodiments, the basic components of the liquid culture medium include: sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, boric acid, manganese chloride tetrahydrate, zinc sulfate, sodium molybdate, copper sulfate pentahydrate, and cobalt chloride hexahydrate.
[0043] In this application, no limitation is made on the mutagenesis method of Chlorella proteoglycans. Those skilled in the art can choose from the well-known methods, such as physical mutagenesis, chemical mutagenesis, biological mutagenesis, etc.
[0044] In some preferred embodiments, physical mutagenesis is employed, using an ambient pressure, room temperature plasma mutagenesis instrument.
[0045] In this application, the concentration of the algal solution encapsulated in microdroplets is 3.5 × 10⁻⁶. 5 —4×10 5 CFU / mL. For example, the concentration of the algal solution can be 3.5 × 10⁻⁶ CFU / mL. 5 CFU / mL, 3.6×10 5 CFU / mL, 3.7×10 5 CFU / mL, 3.8×10 5 CFU / mL, 3.9×10 5 CFU / mL, 4.0×10 5 CFU / mL, or any range thereof.
[0046] In some preferred embodiments, the concentration of the algal solution is 3.5 × 10⁻⁶. 5 CFU / mL or 4.0×10 5CFU / mL.
[0047] In this application, "CFU / mL" represents the number of algae capable of growth and reproduction per milliliter of sample, which is used to determine the activation effect of Chlorella proteoglycans.
[0048] In this application, the method for detecting the concentration (CFU / mL) of Chlorella proteoglycans is not limited. Those skilled in the art can choose from known methods, such as plate coating, pour plate method, membrane filtration, etc. They can also choose other ways to express the concentration of algal solution, such as OD value, cell number / mL, etc., as long as the corresponding algal solution concentration can be achieved.
[0049] In this application, the droplet microfluidic system used to encapsulate the algal solution in microdroplets is not limited. Those skilled in the art can select from known devices, as long as the selected droplet microfluidic system can generate, manipulate, and control microdroplets encapsulated in an immiscible carrier fluid. Furthermore, this application is not limited to the use of a droplet microfluidic system; any device or apparatus that can implement the screening method defined in this application may be used.
[0050] In this application, the diameter of the microdroplets is 70-80 μm. For example, the diameter can be 70 μm, 71 μm, 72 μm, 73 μm, 74 μm, 75 μm, 76 μm, 77 μm, 78 μm, 79 μm, 80 μm, or any range thereof.
[0051] In some preferred embodiments, the diameter of the microdroplets is 70 μm or 80 μm. In some more preferred embodiments, the diameter of the microdroplets is 80 μm.
[0052] In this application, the microdroplets are droplets with a volume of 1-999 picoliters.
[0053] For example, the microdroplets are 1 picoliter, 2 picoliters, 3 picoliters, 4 picoliters, 5 picoliters, 6 picoliters, 7 picoliters, 8 picoliters, 9 picoliters, 10 picoliters, 20 picoliters, 30 picoliters, 40 picoliters, 50 picoliters, 60 picoliters, 70 picoliters, 80 picoliters, 90 picoliters, 100 picoliters, 110 picoliters, 120 picoliters, 130 picoliters, 140 picoliters, 150 picoliters, 160 picoliters, 170 picoliters, 180 picoliters, 190 picoliters, 200 picoliters, 210 picoliters, 220 picoliters, 230 picoliters, 240 picoliters, 250 picoliters, 260 picoliters, 270 picoliters, 280 picoliters, 290 picoliters, 300 picoliters, 310 picoliters, 320 picoliters, 330 picoliters, and 340 picoliters. 350 picoliters, 360 picoliters, 370 picoliters, 380 picoliters, 390 picoliters, 400 picoliters, 410 picoliters, 420 picoliters, 430 picoliters, 440 picoliters, 450 picoliters, 460 picoliters, 470 picoliters, 480 picoliters, 490 picoliters, 500 picoliters, 600 picoliters, 700 picoliters, 800 picoliters, 900 picoliters, 910 picoliters, 920 picoliters, 930 picoliters, 940 picoliters, 950 picoliters, 960 picoliters, 970 picoliters, 980 picoliters, 990 picoliters, 991 picoliters, 992 picoliters, 993 picoliters, 994 picoliters, 995 picoliters, 996 picoliters, 997 picoliters, 998 picoliters, 999 picoliters, etc., or any range thereof.
[0054] In this application, the given conditions include: culturing the encapsulated microdroplets for a given time under certain light conditions and gas pressure, wherein the light conditions are between 200,000 lux and 800,000 lux. For example, the light conditions can be 200,000 lux, 250,000 lux, 300,000 lux, 350,000 lux, 400,000 lux, 450,000 lux, 500,000 lux, 550,000 lux, 600,000 lux, 650,000 lux, 700,000 lux, 750,000 lux, 800,000 lux, or any range thereof.
[0055] In some preferred embodiments, the illumination conditions are between 200,000 lux and 400,000 lux.
[0056] The gas pressure is from 0.01 MPa to 0.04 MPa, for example, it can be 0.01 MPa, 0.02 MPa, 0.03 MPa, 0.04 MPa, or any range therebetween.
[0057] In some preferred embodiments, the gas pressure is 0.01 to 0.02 MPa.
[0058] The cultivation period is defined as the cultivation time until the fluorescence signals generated by different microdroplets can be distinguished by a fluorescence detection device.
[0059] In this application, the fluorescence signal detection device is not limited, and those skilled in the art can select it according to common knowledge, such as a fluorescence microplate reader, fluorescence microscope, fluorescence spectrophotometer, microplate reader, flow cytometer, laser confocal microscope, etc.
[0060] In this application, the implementation of the given conditions (light conditions, gas pressure) is not limited. Those skilled in the art can select some devices or instruments based on common knowledge, as long as they can achieve the light conditions and gas pressure specified in this application.
[0061] In some preferred embodiments, the given conditions are achieved by the above-described device, which can collect the microdroplets in a conduit, wind the conduit around a coil rack, place the coil rack in the device space, and cultivate the microdroplets by adjusting the gas flow rate and the light source.
[0062] In some preferred embodiments, the conduit is a Teflon conduit; the gas flow rate is achieved by connecting a gas cylinder; and the light source is achieved by installing a planar light source.
[0063] This application further provides the use of the device in culturing microdroplets.
[0064] In this application, the microdroplets are the microdroplets encapsulated in the above method.
[0065] The method for screening Chlorella proteoglycans mutants provided in this application involves encapsulating the mutated Chlorella proteoglycan slurry in microdroplets and culturing it under certain conditions. The Chlorella proteoglycans are then screened by detecting the fluorescence signal of the cultured microdroplets. Compared with existing technologies, this method has high throughput, high accuracy, and low cost, and can quickly screen out Chlorella proteoglycan mutants with high growth performance.
[0066] Example This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.
[0067] The procedure for high-throughput screening of rapidly growing Chlorella vulgaris mutants is as follows: Figure 1 As shown.
[0068] Example 1: Activation and culture of Chlorella proteoglycans Take 1 mL of Chlorella proteoglycans culture and transfer it to a shake flask containing 50 mL of liquid culture medium. Incubate at 30°C, a light-dark ratio of 16:8, and a light intensity of 40,000 lux. After multiple subculturings, its growth viability stabilizes. Once Chlorella proteoglycans reaches the logarithmic growth phase, inoculate it at a rate of 5%-10% into fresh liquid culture medium to serve as a seed stock for plasma mutagenesis experiments at ambient pressure and room temperature.
[0069] The basic components of the liquid culture medium include: sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, disodium EDTA, boric acid, manganese chloride tetrahydrate, zinc sulfate, sodium molybdate, copper sulfate pentahydrate, cobalt chloride hexahydrate, and autoclave at 121°C.
[0070] Example 2: Construction of a Chlorella mutant library with protein nucleus Take 1 mL of thoroughly mixed Chlorella proteoglycans seed stock solution, centrifuge at 5000 rpm for 5 min, discard the supernatant, wash 2-3 times with sterile deionized water, and resuspend in 5% glycerol. Mutagenesis of Chlorella proteoglycans was performed using an ambient pressure room temperature plasma mutagenizer (ARTP, Tianmu Biotechnology Co., Ltd.). 10 μL of Chlorella proteoglycans suspension was spread onto a slide, placed on the ARTP stage, and an EP tube containing 1 mL of physiological saline was placed below the stage. The mutagenesis power was set to 120 W, and the mutagenesis time was 15 s. Mutagenesis was initiated, and after completion, the algal solution on the slide was eluted into the physiological saline in the EP tube.
[0071] Example 3: Preparation of Single-Cell Droplets The cells were counted using a hemocytometer, and the mutagenized Chlorella proteoglycan solution was diluted to 3.5 × 10⁻⁶ using liquid culture medium. 5 -4×10 5 Using the algal solution as the aqueous phase and fluorinated oil (droplet preparation oil, Tianmu Biotechnology Co., Ltd.) as the oil phase, water-in-oil droplets with a volume of 270 pL were generated at a rate of 2000-3000 droplets / s using the droplet preparation function of the droplet microfluidic cell sorter (DREM cell, Tianmu Biotechnology Co., Ltd.).
[0072] Example 4: Optimization of Algal Solution Concentration In droplet microfluidic systems, the distribution of the number of cells encapsulated within droplets follows the Poisson distribution law. Taking a droplet volume of 270 pL as an example, the single-cell encapsulation rate for the same droplet volume with different cell concentrations is shown in Table 1. Table 1 shows that when the algal solution concentration is 3.5 × 10⁻⁶ pL... 5 —4×10 5At CFU / mL, the single-cell encapsulation rate is close to 10%, while the multi-cell encapsulation rate is controlled at an extremely low level, meeting the optimal conditions for single-cell screening. Below this range, the empty droplet rate is too high, resulting in extremely low screening throughput and rendering it impractical. Above this range, the multi-cell encapsulation rate increases significantly. During screening, these droplets, due to their stronger fluorescence signal, may be misidentified as rapidly growing algal cells, leading to a sharp increase in the false positive rate and severely compromising the accuracy of screening. See [Figure showing droplet encapsulation at different concentrations] for details. Figure 2 .
[0073] In summary, the final value was determined to be 3.5 × 10⁻⁶. 5 -4×10 5 The optimal concentration range of CFU / mL ensures sufficient single-cell encapsulation for high throughput while minimizing multi-cell encapsulation, thus guaranteeing high accuracy of growth rate screening results based on fluorescence signals.
[0074] Table 1. Effect of different algal solution concentrations on single-cell droplet encapsulation efficiency
[0075] Example 5: Optimization of droplet diameter To optimize the single-cell screening process, this example investigated the effects of different droplet diameters on the single-cell encapsulation efficiency and subsequent culture stability of *Chlorella proteoglycans*. The mutagenized algal solution (concentration 3.75 × 10⁻⁶) was used. 5 Microdroplets of different diameters (CFU / mL) were prepared for comparative testing.
[0076] The results showed that droplet size is a key factor affecting encapsulation efficiency and operational feasibility. When the droplet diameter is too small (60 μm), the single-cell encapsulation rate is low, and the limited physical space within the droplet severely restricts the normal growth and proliferation of algal cells. When the droplet diameter increases to the range of 70-80 μm, the single-cell encapsulation rate tends to be ideal, and the droplets exhibit good stability during culture and manipulation. When the droplet diameter further increases (90 μm and above), although the encapsulation rate improves slightly, its structural stability decreases, making it more prone to breakage or fusion during flow and culture. Simultaneously, because droplets are three-dimensional structures, larger droplets lead to increased depth of field under the microscope, making it difficult to focus and observe and assess the true state of all cells, significantly reducing detection efficiency. Furthermore, large droplets directly reduce the number of droplets per unit volume, resulting in a significant decrease in overall screening throughput. A comparison of the encapsulation effects of droplets with different diameters is provided. Figure 3 As shown.
[0077] In summary, droplets with a diameter of approximately 70-80 μm, especially those with a diameter of 80 μm (volume of approximately 270 pL), achieve the best balance between single-cell encapsulation efficiency, droplet stability, ease of observation, and screening throughput, and are therefore determined to be the optimal size for implementing this screening method.
[0078] Example 6: Optimization of Droplet Culture Conditions Microalgae growth requires the accumulation of matter and energy through photosynthesis. For the cultivation of microalgae within picoliter-sized microdroplets, several key issues need to be addressed: 1. Uniform and sufficient light intensity must be provided between the droplets; 2. Sufficient gas exchange must exist between the droplets and the external environment; 3. Evaporation of the droplets must be prevented during cultivation. To address these issues, this application designs a dedicated droplet cultivation device, see [link to details]. Figure 4 .
[0079] Droplets are collected in a highly permeable and light-transmitting Teflon conduit, which is then evenly wound around a coil rack to ensure uniform illumination between the droplets. The coil rack is placed in a sealed space with small openings on the left and right sides as gas inlets and outlets. A 4mm gas tube is fixed at the left inlet and connected to a 40L gas cylinder containing 5% CO2. The gas flow rate is regulated by a pressure reducing valve connected to the gas cylinder. To accelerate air circulation, an electric fan is added to the bottom of the device, while simultaneously reducing the heat radiation from the light source. A planar light source with adjustable brightness and illumination period is installed on the upper part of the device.
[0080] The illumination was adjusted to 5000 lux, 10000 lux, 20000 lux, 50000 lux, 100000 lux, 200000 lux, 400000 lux, 800000 lux, 1200000 lux, and 1600000 lux, respectively, with a light / dark cycle of 16 / 8 h. 5% CO2 gas was introduced at a pressure of 0.02 MPa. The droplets were cultured for 48 h, and the droplets were placed in a cell counting chamber. The average number of Chlorella proteoglycans (n>10) within the droplets was observed and counted under a fluorescence microscope. The results are shown in Table 2 below.
[0081] Table 2 Effects of different light intensities on microalgal growth
[0082] Table 2 shows that different light intensities have a significant impact on the growth of microalgae. When the light intensity is below 20,000 lux, the growth of microalgae is significantly inhibited, meaning that the light intensity is too low to provide sufficient energy for photosynthesis, leading to growth stagnation. When the light intensity exceeds 200,000 lux, the growth of microalgae accelerates significantly, gradually stabilizing at 800,000 lux. When the light intensity exceeds 800,000 lux, a significant photoinhibition phenomenon occurs, with the average number of microalgae decreasing instead of increasing at 1,200,000 lux. When the light intensity reaches 1,600,000 lux, the number of algal cells decreases significantly, which is a typical manifestation of photosynthetic system damage and chlorophyll decomposition. This indicates that excessively high light intensity can cause photodamage to *Chlorella pyrenoidosa*, thus inhibiting its growth.
[0083] The pressure of the introduced 5% CO2 gas was adjusted to 0 MPa, 0.01 MPa, 0.02 MPa, and 0.04 MPa, respectively. The illumination intensity was 40,000 lux, and the light / dark cycle was 16 / 8 h. The droplets were cultured for 48 h. The droplets were then placed in a Countstar cell counting chamber, and the average number of Chlorella proteoglycans in the droplets (n>10) was observed and counted under a fluorescence microscope. The results are shown in Table 3 below.
[0084] Table 3 Effects of different gas flow rates on microalgae growth
[0085] Table 3 shows that different gas flow rates have a significant impact on the growth of microalgae; when the gas cylinder output pressure is <0.01 MPa, the growth of microalgae is significantly inhibited; when the gas flow rate is ≥0.02 MPa, the growth of microalgae gradually tends to stabilize.
[0086] Based on the screening results for light intensity and gas flow rate, droplets were cultured under the following conditions: light intensity of 40,000 lux, light / dark cycle of 16 / 8 h, and 5% CO2 gas at a pressure of 0.02 MPa. Droplets were collected at 0 h, 12 h, 24 h, 36 h, and 48 h and placed in a cell counting chamber. The number of *Chlorella proteoglycans* within the droplets was observed and counted under a fluorescence microscope, and fluorescence was collected. The results are as follows: Figure 6 As shown, *Chlorella proteoglycans* begins to grow within the droplet during 12-24 hours, proliferates rapidly during 24-36 hours, and quickly fills the droplet during 36-48 hours. Furthermore, the microalgae are green and rich in chlorophyll; chlorophyll itself exhibits a characteristic emission peak at a wavelength of 680 nm, such as... Figure 5 As shown, the intensity of red fluorescence in the droplet increases linearly with the increase in the number of Chlorella proteoglycans in the droplet. Therefore, the fluorescence intensity in the droplet can be used to characterize the number of Chlorella proteoglycans in the droplet, thereby reflecting the growth status of Chlorella proteoglycans.
[0087] Example 7 Droplet Screening Using a DREM cell, droplets cultured for 48 hours were sampled and screened at a rate of 500-600 droplets / s. The excitation wavelength was set to 475 nm, the detection band to 670 / 20 nm, the laser power to 20 mW, the electric field voltage to 800 V, and the sorting time to 200 μs. The real-time signal of the *Chlorella proteoglycans* droplets was as follows: Figure 7 As shown, by Figure 7 It can be seen that different droplets produced fluorescence signals of different intensities. The intensity of these signals is positively correlated with the biomass of algal cells (i.e., chlorophyll content) within the droplets, directly reflecting the differences in growth rates among different mutant algal strains.
[0088] Fluorescence signals within the droplets were collected and histogram analysis was performed. The results are as follows: Figure 8 As shown, the droplet with the highest fluorescence signal (1%) was selected as the target droplet for sorting. After 1 hour of sorting, 10,000 droplets were obtained from 2,000,000 droplets. The sorting results are shown below. Figure 9 As shown, by Figure 9 It can be seen that the number of algal cells inside the sorted droplets (right side) is significantly higher than that of the original droplet population (left side) before sorting, confirming that this method successfully enriched the Chlorella proteoglycan mutant that achieved a higher degree of proliferation in the droplets, i.e., the target algal strain with a faster growth rate.
[0089] 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. An apparatus, characterized in that, The culture device includes an upper layer and a lower layer. The upper layer has a light source and a coil rack at the top, and air inlets and outlets on both sides of the coil rack. Preferably, the air inlet is connected to a gas cylinder, and the flow rate of the incoming gas is adjusted by a pressure reducing valve connected to the gas cylinder.
2. The apparatus according to claim 1, characterized in that, A fan is installed at the bottom of the lower layer.
3. A method for screening protein-nucleated Chlorella mutants, characterized in that, Includes the following steps: Chlorella pyrenoidosa was prepared by activating, culturing and mutagenesis to obtain Chlorella pyrenoidosa algal solution; The mutagenized algal solution was encapsulated in microdroplets; Under given conditions, the encapsulated microdroplets are cultured for a given time. The fluorescence signal of microdroplets after culture was detected, and different growth rates of Chlorella proteoglycans were screened based on the intensity of the fluorescence signal.
4. The method according to claim 3, characterized in that, The concentration of the mutagenic Chlorella proteoglycan solution was 3.5 × 10⁻⁶. 5 —4×10 5 CFU / mL.
5. The method according to claim 3, characterized in that, The diameter of the microdroplets is 70-80 μm, preferably 80 μm.
6. The method according to claim 3, characterized in that, The microdroplets are 1-999 picoliters in size.
7. The method according to claim 3, characterized in that, The given conditions include: culturing the encapsulated microdroplets under certain light conditions and gas pressure for a given time. The illumination conditions are 200,000 lux to 800,000 lux; preferably 200,000 lux to 400,000 lux. The gas pressure is 0.01 MPa to 0.04 MPa; preferably 0.01 to 0.02 MPa. The given time is for cultivation until the fluorescence signals generated by different microdroplets can be distinguished by a fluorescence detection device.
8. The method according to claim 7, characterized in that, The given conditions are achieved by the apparatus of any one of claims 1 or 2.
9. Use of the device according to claim 1 or 2 in the cultivation of microdroplets.
10. The use according to claim 9, characterized in that, The microdroplets are the encapsulated microdroplets as described in any one of claims 3 to 6.