A rapid screening method for chlorella mutagenic strains

Abstract

The present application relates to the field of microbial technology, and discloses a rapid screening method for Chlorella mutagenesis strains. The method comprises: (1) mutagenesis treatment of Chlorella algae seed to obtain mutagenesis algae group; (2) at least three rounds of progressive screening of the mutagenesis algae group to obtain high-performance Chlorella; wherein the first round of screening is carried out in a microplate, growth rate is used as the screening index, and the algae strains with growth rate ranking from the top 10% to the top 20% are selected; the second round of screening is carried out in a deep well plate, growth rate is used as the screening index, and the algae strains with growth rate ranking from the top 10% to the top 20% are selected; the third round of screening is carried out in a conical flask, growth rate, biomass and protein content are used as the comprehensive screening index, and high-performance Chlorella is obtained. The high-throughput screening method provided by the present application has the advantages of high efficiency, universality and reliability, can significantly shorten the breeding cycle, and can stably screen high-performance Chlorella strains from different mutagenesis populations.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and specifically to a rapid screening method for Chlorella mutant strains. Background Technology

[0002] Improving the traits of microalgae (especially Chlorella) using mutagenesis breeding techniques (such as increasing growth rate, biomass, and content of specific products) is an important research direction in the field of biotechnology. Whether it is physical mutagenesis (such as gamma rays and ultraviolet light) or chemical mutagenesis (such as EMS), one of the core challenges is how to quickly and accurately identify the very few superior individuals whose performance has undergone positive leaps from thousands of mutants.

[0003] Currently, common screening strategies mainly fall into two categories, but both have significant limitations: Firstly, there is the phenotypic direct screening method based on final performance. This method typically involves multiple rounds of expansion culture of the mutagenized bacterial population and direct measurement of final industrial traits such as biomass, lipid content, and protein content. While this method yields reliable results, it is usually extremely time-consuming, labor-intensive, low-throughput, and costly. More importantly, the entire screening process is inefficient and difficult to systematize or platformize.

[0004] Secondly, indirect prediction and screening methods based on physiological and biochemical indicators: To accelerate the screening process, existing technologies have developed methods to indirectly predict strain growth performance by measuring certain physiological surrogate indicators (such as photosynthetic oxygen evolution rate, chlorophyll fluorescence parameters, etc.). While these methods are faster, they have drawbacks: The accuracy of prediction needs improvement: the correlation between surrogate indicators and final industrial traits (such as biomass) is not absolute, potentially leading to false positives or false negatives; Limited universality: many surrogate indicators only apply to specific metabolic pathways (such as photoautotrophy), and may no longer be applicable when the target trait or culture mode changes (such as screening for high-yield strains for heterotrophic fermentation); Lack of predictability for industrial scale-up: strains performing well in micro-systems may fail when scaled up to reactor scale due to environmental adaptability issues, and simple indicator detection cannot predict this.

[0005] In summary, there is an urgent need in this field for a universal screening technology platform that can overcome the aforementioned shortcomings. This platform should possess the following characteristics: high throughput and high efficiency, significantly shortening the screening cycle; standardized and reproducible screening procedures, independent of operator experience; applicability to multiple mutagen sources and different target traits; and early assessment of strain stability and industrial scale-up potential during the screening process. However, currently, no systematic screening method has been reported that simultaneously meets all of the above requirements. Summary of the Invention

[0006] The present invention aims to overcome the problems of low predictive accuracy, limited versatility, and lack of foresight for industrial scale-up in the screening of microalgae improved by mutation breeding technology in existing technologies.

[0007] To achieve the above objectives, the present invention provides a rapid screening method for Chlorella mutant strains, the method comprising the following steps: (1) Mutagenesis was performed on Chlorella species to obtain a mutant algal community; (2) Perform at least three rounds of progressive screening on the mutagenic algal community to obtain high-performance Chlorella; The first round of screening was carried out in microplates, with growth rate as the screening index, selecting the top 10% to top 20% of algal strains in terms of growth rate. The second round of screening was carried out in deep well plates, using growth rate as the screening index, and selecting the top 10% to top 20% of algal strains in terms of growth rate. The third round of screening was conducted in conical flasks, using growth rate, biomass, and protein content as comprehensive screening indicators. Algal strains that simultaneously met the criteria of growth rate ≥ 150% of wild-type strains, biomass ≥ 200% of wild-type strains, and protein content ≥ 120% of wild-type strains were selected as qualified algal strains. Then, among the qualified algal strains, they were sorted from high to low biomass, and the top 10% to top 20% of algal strains in terms of biomass were selected as the final high-performance Chlorella.

[0008] Compared with existing technologies, the method provided by this invention has at least the following beneficial effects: 1. The method provided by this invention has excellent versatility and adaptability, and has constructed a technical platform that does not depend on a specific mutagenesis source. Whether it is physical gamma ray or chemical EMS mutagenesis, it can be seamlessly connected with the subsequent standardized screening process through its core "lethality rate > 90%" dose determination principle. It has been successfully applied to a variety of economic microalgae, which significantly improves the universality and success rate of breeding work. 2. The method provided by this invention has a high degree of systematicity and foresight. The pioneering three-stage progressive screening system of "microplate-deep plate-conical flask" simulates the industrial scale-up process and combines multiple indicators such as growth rate, biomass, and protein content for comprehensive evaluation. It can eliminate strains unsuitable for scale-up at an early stage and accurately identify elite algal strains with the best overall performance and industrialization potential, which greatly reduces the risk of later development. 3. The method provided by this invention achieves reproducibility and high efficiency. By parameterizing and standardizing key steps, this scheme ensures a high degree of repeatability in the screening process. Simultaneously, by utilizing high-throughput microplate technology to process massive numbers of mutants in parallel, the traditional screening cycle of several months is significantly shortened to several weeks. Its superior efficiency has been empirically demonstrated by screening high-performance strains with biomass increases of approximately 2.94 times and 2.5 times that of wild-type strains, respectively, making it a reliable breeding platform capable of continuous production. Attached Figure Description

[0009] Figure 1 These are photographs of the growth of Chlorella after treatment with different irradiation doses in a preferred embodiment of the present invention. Figure 2 The OD of Chlorella after treatment with different irradiation doses in a preferred embodiment of the present invention 680 value; Figure 3 This is the lethality rate of Chlorella after treatment with different irradiation doses in a preferred embodiment of the present invention; Figure 4 The OD of high-performance Chlorella obtained in a preferred embodiment of the present invention 680 Value diagram; Figure 5 This is a comparison chart of the biomass of high-performance Chlorella obtained in a preferred embodiment of the present invention; Figure 6 This is a comparison chart of protein concentrations in high-performance Chlorella strains obtained in a preferred embodiment of the present invention. Figure 7 This is a flowchart of a three-round progressive screening of the mutagenized algal community after mutagenesis treatment in a preferred embodiment of the present invention; Figure 8 These are photographs of Chlorella growth after treatment with different doses of ethyl methanesulfonate in a preferred embodiment of the present invention. Figure 9 The OD of Chlorella vulgaris treated with different doses of ethyl methanesulfonate in a preferred embodiment of the present invention is... 680 value; Figure 10 This is the lethality rate of Chlorella after treatment with different doses of ethyl methanesulfonate in a preferred embodiment of the present invention; Figure 11 This is another preferred embodiment of the present invention that yields high-performance Chlorella OD. 680 Value diagram; Figure 12 This is a comparison chart of the biomass of high-performance Chlorella obtained in another preferred embodiment of the present invention; Figure 13This is a comparison chart of protein concentrations of high-performance Chlorella obtained in another preferred embodiment of the present invention; Figure 14 These are photographs of the growth status of the high-performance Chlorella selected in Example 1 of Test Example 1 of the present invention; Figure 15 These are photos of the growth status of the high-performance Chlorella selected in Example 2 of Test Example 1 of this invention; Figure 16 This is a biomass test diagram of the high-performance Chlorella screened in Example 1 of the present invention; Figure 17 This is a biomass test diagram of the high-performance Chlorella screened in Example 2 of Test Example 1 of the present invention. Detailed Implementation

[0010] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0011] As mentioned above, this invention provides a rapid screening method for Chlorella mutant strains, which includes the following steps: (1) Mutagenesis was performed on Chlorella species to obtain a mutant algal community; (2) Perform at least three rounds of progressive screening on the mutagenic algal community to obtain high-performance Chlorella; The first round of screening was carried out in microplates, with growth rate as the screening index, selecting the top 10% to top 20% of algal strains in terms of growth rate. The second round of screening was carried out in deep well plates, using growth rate as the screening index, and selecting the top 10% to top 20% of algal strains in terms of growth rate. The third round of screening was conducted in conical flasks, using growth rate, biomass, and protein content as comprehensive screening indicators. Algal strains that simultaneously met the criteria of growth rate ≥ 150% of wild-type strains, biomass ≥ 200% of wild-type strains, and protein content ≥ 120% of wild-type strains were selected as qualified algal strains. Then, among the qualified algal strains, they were sorted from high to low biomass, and the top 10% to top 20% of algal strains in terms of biomass were selected as the final high-performance Chlorella.

[0012] Preferably, the method of the present invention further includes pretreating the Chlorella strain before performing the mutagenesis treatment, including: inoculating the Chlorella strain into BG-11 liquid medium, culturing it at 25±2℃, light intensity of 2000-5000 lux, and a 12-hour light-dark cycle until the logarithmic growth phase, collecting the algal cells by centrifugation, washing them 2-3 times with sterile physiological saline, and preparing a solution with a concentration of 10... 6 -10 8 A uniform Chlorella suspension with cells / mL was then subjected to the aforementioned mutagenesis treatment.

[0013] More preferably, the criterion for determining the logarithmic growth phase is OD. 680 It is 0.6-0.8.

[0014] In a preferred embodiment, the mutagenesis treatment is physical mutagenesis and / or chemical mutagenesis.

[0015] Preferably, the method of the present invention further includes, after completing the mutagenesis treatment, using the algal solution treated with the lowest dose group with a lethality rate of 90% or higher as the mutagenized algal population.

[0016] More preferably, the physical mutagenesis includes gamma-ray irradiation, and the chemical mutagenesis includes alkylating agent treatment.

[0017] According to a preferred embodiment, the physical mutation method is as follows: 60 Co-γ irradiation, with an irradiation dose of 400-2000 Gy.

[0018] In a preferred embodiment, the chemical induction is performed with ethyl methanesulfonate at a concentration of 0.6-1.0 wt%.

[0019] Preferably, when using the above 60 During Co-γ ray irradiation, the mutagenesis treatment includes the following operations: choose 60 Using Co-γ as the ray source, irradiation doses of 0 Gy, 400 Gy, 800 Gy, 1200 Gy, 1600 Gy, and 2000 Gy were set to irradiate Chlorella algal solutions. The solutions were suspended in sterile water, diluted, and 50 μL of the liquid was spread onto BG-11 solid medium containing 10 g / L glucose. The solutions were cultured in the dark at 25°C for 7 days. The number of single colonies was counted, and the lethality rate under different irradiation doses was calculated. The algal solutions irradiated with the lowest irradiation dose group with a lethality rate of over 90% were selected as the mutagenic algal community.

[0020] Preferably, when treated with the ethyl methanesulfonate, the mutagenesis treatment includes the following steps: Suspended Chlorella solutions containing 0 wt%, 0.2 wt%, 0.4 wt%, 0.6 wt%, 0.8 wt%, and 1.0 wt% ethyl methanesulfonate were prepared and chemically mutagenized for 20 min. The solutions were then suspended in sterile water, diluted, and 50 μL of the liquid was spread onto BG-11 solid medium containing 10 g / L glucose. The solutions were incubated in the dark at 25°C for 7 days. The number of single colonies was counted, and the lethality rate under different chemical mutagenization treatment doses was calculated. The mutagenized algal solution with the lowest ethyl methanesulfonate dose group having a lethality rate of 90% or higher was taken as the mutagenized algal population.

[0021] According to a preferred embodiment, in step (2), the first round of screening includes: inoculating the mutagenic algal community into a 96-well microplate containing 10 g / L glucose in BG-11 liquid medium, culturing it in the dark at 25°C for 7 days, and then measuring the OD of each well. 680 Then, fast-growing algal strains are screened based on their growth rate. Under this optimal condition, it is helpful to quickly screen dominant algal strains from a large number of mutant strains and eliminate low-performing algal strains.

[0022] In a preferred embodiment, the BG-11 liquid culture medium comprises the following components: 1.02 g / L sodium nitrate, 0.1145 g / L dipotassium hydrogen phosphate, 0.15 g / L magnesium sulfate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.06 g / L citric acid, 0.06 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, and 1 mL / L A5 solution.

[0023] Preferably, the BG-11 liquid culture medium containing 10 g / L glucose is a culture medium obtained by adding an additional 10 g / L of glucose to the BG-11 liquid culture medium.

[0024] Preferably, the BG-11 solid culture medium containing 10 g / L glucose is a culture medium obtained by adding an additional 10 g / L glucose and 15 g / L agar to the BG-11 liquid culture medium.

[0025] Preferably, in this invention, the A5 solution is a solution containing the following concentrations and components: 2.86 g / L boric acid, 1.81 g / L manganese chloride monohydrate, 0.222 g / L zinc sulfate heptahydrate, 0.079 g / L copper sulfate pentahydrate, 0.390 g / L sodium molybdate dihydrate, and 0.049 g / L cobalt nitrate hexahydrate.

[0026] Preferably, in step (2), the screening index based on growth rate includes: measuring the OD of the microplate in each well at the beginning and end of the culture. 680 The average growth rate is calculated based on the value, and then further screening is performed.

[0027] In a preferred embodiment, in step (2), the second round of screening includes: transferring the algal strains selected in the first round to a 96-well plate with a system volume of 1 mL, culturing them in the dark at 25°C for 7 days, and then measuring the OD of each well. 680 Then, the growth rate was used as an indicator to screen for algal strains that grow rapidly and continuously.

[0028] In a preferred embodiment, in step (2), the third round of screening includes: inoculating the algal strains selected in the second round into conical flasks for heterotrophic culture for 10 days, and measuring the OD after the culture is completed. 680 High-performance Chlorella were screened using growth rate, biomass, and protein content as indicators.

[0029] In a preferred embodiment, the heterotrophic culture medium comprises the following components: 0.1145 g / L dipotassium hydrogen phosphate, 0.15 g / L magnesium sulfate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.06 g / L citric acid, 0.06 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, and 1 mL / L A5 solution, with the addition of 10 g / L glucose and 3.75 g / L sodium nitrate.

[0030] In a preferred embodiment, the heterotrophic culture is carried out in a constant temperature shaking incubator in a shaker, and at least the following conditions are met: the temperature is 25°C and the shaking speed is 120 rpm.

[0031] The present invention will be described in detail below through examples. Unless otherwise specified, the raw materials used are all commercially available products.

[0032] BG-11 liquid medium containing 10 g / L glucose: contains 1.02 g / L sodium nitrate, 0.1145 g / L dipotassium hydrogen phosphate, 0.15 g / L magnesium sulfate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.06 g / L citric acid, 0.06 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, and 1 mL / L A5 solution, with an additional 10 g / L glucose.

[0033] The heterotrophic culture medium contains 0.1145 g / L dipotassium hydrogen phosphate, 0.15 g / L magnesium sulfate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.06 g / L citric acid, 0.06 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, and 1 mL / L A5 solution, with additional additions of 10 g / L glucose and 3.75 g / L sodium nitrate.

[0034] The lethality rate was determined by counting the number of single colonies using the plate count method, and calculated using the following formula: Lethality = (Number of single colonies in control group - Number of single colonies in mutant group) / Number of single colonies in control group × 100%; The control group consisted of algal solution with an irradiation dose of 0 Gy or a suspended algal solution with a methanesulfonate concentration of 0 wt%.

[0035] Pretreatment of Chlorella strains: The *Chlorella* strain was inoculated into BG-11 liquid medium and cultured at 25±2℃, light intensity of 2000-5000 lux, and a 12-hour light-dark cycle until the logarithmic growth phase (OD2). 680 (0.6-0.8%), collect algal cells by centrifugation, wash 2-3 times with sterile physiological saline, and prepare a solution with a concentration of 10. 6 -10 8 A uniform suspension of Chlorella cells / mL. Example 1

[0036] This embodiment illustrates that the rapid screening method for Chlorella mutant strains provided by the present invention is performed according to the following steps: This embodiment adopts 60 Physical mutagenesis induced by Co-γ ray irradiation; (1) Choose 60 Using Co-γ as the ray source, irradiation doses of 0 Gy (CON), 400 Gy, 800 Gy, 1200 Gy, 1600 Gy, and 2000 Gy were set. The Chlorella suspension obtained by irradiation pretreatment was suspended in sterile water, diluted, and 50 μL of the liquid was spread on BG-11 solid medium containing 10 g / L glucose (i.e., 10 g / L glucose and 15 g / L agar were added to BG-11 liquid medium). The culture was carried out in the dark at 25°C for 7 days, and the growth was recorded. The number of single colonies was counted by plate counting, and the lethality rate under different irradiation doses was calculated. The algal solution after irradiation with the lowest irradiation dose group (1200 Gy) with a lethality rate of more than 90% was used as the mutagenic algal community. in, Figure 1 The growth of Chlorella after treatment with different irradiation doses is shown; Figure 2 and Figure 3 The OD values ​​of Chlorella after treatment with different irradiation doses are shown. 680 Values ​​and concentrations; (2) The mutant algal community was subjected to a three-round progressive screening to obtain high-performance Chlorella: The first round of screening was conducted in microplates, using growth rate as the screening metric. The induced algal community was inoculated into 96-well microplates with a working volume of 200 μL in BG-11 liquid medium containing 10 g / L glucose. After incubation at 25°C in the dark for 7 days, the OD of each well was measured. 680 The OD values ​​of the microplate in each well were measured at the beginning and end of the culture. 680 The average growth rate was calculated, and the algal strains with the highest growth rates (top 10% to top 20%) were selected. The second round of screening was conducted in a deep-hole plate, using growth rate as the screening criterion: The algal strains selected in the first round of screening were transferred to 96-well plates with a working volume of 1 mL and cultured in the dark at 25°C for 7 days. The OD values ​​of each well were then measured. 680 The OD values ​​of the microplate at the beginning and end of the culture were measured. 680 The average growth rate was calculated, and the top 10% to top 20% of algal strains in terms of growth rate were selected, then transferred and preserved to construct a mutant library. The third round of screening used growth rate, biomass, and protein content as comprehensive screening indicators to obtain high-performance Chlorella: The mutant library selected in the second round of screening was inoculated into Erlenmeyer flasks and heterotrophically cultured in a shaker incubator at 25°C and 120 rpm in the dark for 7 days. OD was measured after the culture was completed. 680 The algal strains that simultaneously meet the following criteria are selected: growth rate ≥ 150% of wild strains, biomass ≥ 200% of wild strains, and protein content ≥ 120% of wild strains. Then, among the qualified algal strains, the algal strains ranked from high to low biomass are selected from the top 10% to top 20% of biomass as the final high-performance Chlorella, named Y11, Y12, Y17, Y18, Y25, and Y30, respectively. Figure 4 The OD of the high-performance Chlorella obtained in this embodiment is shown. 680 The values ​​are given, where "WT" represents the wild strain. As can be seen from the figure, the growth rate of the high-performance Chlorella obtained in this embodiment is significantly higher than that of the wild strain, with high-performance Chlorella Y25 growing the fastest.

[0037] Figure 5 The figure shows the biomass differences of the high-performance Chlorella obtained in this embodiment, where "WT" represents the wild strain. It can be seen from the figure that the biomass concentration of the high-performance Chlorella obtained in this embodiment is significantly higher than that of the wild strain, with the high-performance Chlorella Y25 having the highest concentration. Figure 6The figure shows the protein concentration differences of the high-performance Chlorella obtained in this embodiment, where "WT" represents the wild strain. It can be seen from the figure that the protein concentration of the high-performance Chlorella obtained in this embodiment is significantly higher than that of the wild strain. Figure 7 The process of performing a three-round progressive screening of the induced algal community after mutagenesis treatment is shown. Example 2

[0038] This embodiment uses a method similar to that of Example 1, except that in step (1), instead of physical mutagenesis, chemical mutagenesis is performed using ethyl methanesulfonate treatment, specifically including: (1) Prepare Chlorella suspensions containing 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% ethyl methanesulfonate by mass and chemically mutagenesis for 20 min. Suspend in sterile water, dilute and spread 50 μL of the liquid onto BG-11 solid medium containing 10 g / L glucose. Incubate at 25°C in the dark for 7 days, record the growth, count the number of single colonies, and calculate the lethality under different chemical mutagenesis treatment doses by plate counting. The mutagenesis solution with the lowest ethyl methanesulfonate dose (0.8 wt%) with a lethality of more than 90% is taken as the mutagenesis algal group. in, Figure 8 The growth of Chlorella was shown after treatment with different doses of ethyl methanesulfonate. Figure 9 and Figure 10 The OD values ​​of Chlorella treated with different doses of ethyl methanesulfonate are shown. 680 Value and mortality rate; Finally, in the third round of screening, the mutant library selected in the second round was inoculated into Erlenmeyer flasks and heterotrophically cultured for 7 days in a constant temperature shaking incubator within a shaker at 25°C and a shaking speed of 120 rpm under dark conditions. OD was measured after the culture was completed. 680 The algal strains that simultaneously meet the following criteria are selected: growth rate ≥ 150% of wild strains, biomass ≥ 200% of wild strains, and protein content ≥ 120% of wild strains. Then, among the qualified algal strains, they are sorted from high to low biomass, and the algal strains with the top 10% to top 20% of biomass are selected as the final high-performance Chlorella, named E2, E3, E4, and E5, respectively.

[0039] Figure 11 The OD of the high-performance Chlorella strain obtained in this embodiment is shown. 680 The values ​​are given, where "WT" represents the wild strain. As can be seen from the figure, the growth rate of the high-performance Chlorella obtained in this embodiment is significantly higher than that of the wild strain, with high-performance Chlorella E4 being the fastest. Figure 12 The figure shows the biomass differences of the high-performance Chlorella strains obtained in this embodiment, where "WT" represents the wild strain. It can be seen from the figure that the biomass concentration of the high-performance Chlorella strains obtained in this embodiment is significantly higher than that of the wild strains, with the high-performance Chlorella strain E4 having the highest concentration. Figure 13 The figure shows the protein concentration differences of the high-performance Chlorella strains obtained in this embodiment, where "WT" represents the wild strain. It can be seen from the figure that the protein concentration of the high-performance Chlorella strains obtained in this embodiment is significantly higher than that of the wild strains, with the high-performance Chlorella strain E4 having the highest concentration.

[0040] Test Example 1 The high-performance Chlorella strains obtained from the above screening were subjected to growth and biomass tests, including: High-performance Chlorella vulgaris and wild-type strains (WT group) were cultured in a constant temperature shaker at 25℃ in the dark for 7 days. The growth status and biomass were recorded on day 0 and day 7.

[0041] Figure 14 The growth status of the high-performance Chlorella screened in Example 1 is shown. 60 The high-performance Chlorella obtained by Co-γ mutagenesis showed significantly better growth performance than the wild strain; Figure 15 The growth status of the high-performance Chlorella screened in Example 2 is shown. It can be seen that the growth performance of the high-performance Chlorella obtained by ethyl methanesulfonate mutagenesis is significantly better than that of the wild strain. Figure 16 The biomass of the high-performance Chlorella Y25 strain screened in Example 1 is shown, where "WT" represents the wild-type strain. 60 The biomass of the high-performance Chlorella obtained by Co-γ mutagenesis was significantly higher than that of the wild strain; Figure 17 The biomass of the high-performance Chlorella E4 strain screened in Example 2 is shown, where "WT" represents the wild strain. It can be seen that the biomass of the high-performance Chlorella induced by ethyl methanesulfonate is significantly higher than that of the wild strain.

[0042] Among the algal strains obtained through physical mutagenesis under the same conditions, Y25 had the highest biomass, reaching 3.85 g / L, which is 2.94 times that of the wild strain (biomass: 1.31 g / L). Among the algal strains obtained through chemical mutagenesis, E4 had the highest biomass, reaching 3.26 g / L, which is 2.49 times that of the wild strain.

[0043] Test Example 2 Genetic and physiological / biochemical tests were performed on the high-performance Chlorella strains obtained from the above examples: Multiple generations of subculturing were performed on strains Y11, Y12, Y17, Y18, Y25, Y30, E2, E3, E4, and E5. The results showed that all strains exhibited stable traits and were capable of stable inheritance. Compared with the wild type, the monoclonal colonies formed by these high-performance Chlorella strains were significantly larger in diameter and darker green in color.

[0044] The results above show that the screening method established by the method provided by this invention has successfully shortened the screening cycle of high-performance algal strains from the traditional several months to about 30 days, and has been successfully applied to different mutagenesis sources such as γ-rays and EMS. Finally, it has efficiently and reproducibly screened high-performance Chlorella strains with a biomass increase of more than 2.5 times, providing an efficient and universal technical platform for Chlorella breeding.

[0045] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A rapid screening method for Chlorella mutant strains, characterized in that, The method includes the following steps: (1) Mutagenesis was performed on Chlorella species to obtain a mutant algal community; (2) Perform at least three rounds of progressive screening on the mutagenic algal community to obtain high-performance Chlorella; The first round of screening was carried out in microplates, with growth rate as the screening index, selecting the top 10% to top 20% of algal strains in terms of growth rate. The second round of screening was carried out in deep well plates, using growth rate as the screening index, and selecting the top 10% to top 20% of algal strains in terms of growth rate. The third round of screening was conducted in conical flasks, using growth rate, biomass, and protein content as comprehensive screening indicators. Algal strains that simultaneously met the criteria of growth rate ≥ 150% of wild-type strains, biomass ≥ 200% of wild-type strains, and protein content ≥ 120% of wild-type strains were selected as qualified algal strains. Then, among the qualified algal strains, they were sorted from high to low biomass, and the top 10% to top 20% of algal strains in terms of biomass were selected as the final high-performance Chlorella.

2. The method according to claim 1, characterized in that, The mutagenesis treatment is physical mutagenesis and / or chemical mutagenesis.

3. The method according to claim 1 or 2, characterized in that, The physical mutagenesis is irradiated with gamma rays; and / or The chemical induction process involves alkylating agent treatment.

4. The method according to claim 3, characterized in that, The physical mutagenesis method is as follows: 60 Co-γ irradiation, with an irradiation dose of 400-2000 Gy; and / or, The chemical induction process involves treatment with ethyl methanesulfonate at a concentration of 0.6-1.0 wt%.

5. The method according to claim 1, characterized in that, In step (2), the first round of screening includes: inoculating the mutagenic algal community into a 96-well microplate containing 10 g / L glucose in BG-11 liquid medium, culturing it in the dark at 25°C for 7 days, and then measuring the OD of each well. 680 Then, fast-growing algal strains are selected based on their growth rate.

6. The method according to claim 5, characterized in that, The BG-11 liquid culture medium containing 10 g / L glucose comprises the following components: 1.02 g / L sodium nitrate, 0.1145 g / L dipotassium hydrogen phosphate, 0.15 g / L magnesium sulfate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.06 g / L citric acid, 0.06 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, and 1 mL / L A5 solution, with an additional 10 g / L glucose.

7. The method according to claim 1, characterized in that, In step (2), the second round of screening includes: transferring the algal strains selected in the first round to a 96-well plate with a system volume of 1 mL, culturing them in the dark at 25°C for 7 days, and then measuring the OD of each well. 680 Then, the growth rate was used as an indicator to screen for algal strains that grow rapidly and continuously.

8. The method according to claim 1, characterized in that, In step (2), the third round of screening specifically involves: inoculating the algal strains selected in the second round into conical flasks for heterotrophic culture for 10 days, and measuring the OD after the culture is completed. 680 High-performance Chlorella were screened using growth rate, biomass, and protein content as indicators.

9. The method according to claim 8, characterized in that, The heterotrophic culture medium contains the following components: 0.1145 g / L dipotassium hydrogen phosphate, 0.15 g / L magnesium sulfate heptahydrate, 0.036 g / L calcium chloride dihydrate, 0.06 g / L citric acid, 0.06 g / L ferric ammonium citrate, 0.001 g / L disodium ethylenediaminetetraacetate, 0.02 g / L sodium carbonate, and 1 mL / L A5 solution, with additional additions of 10 g / L glucose and 3.75 g / L sodium nitrate.

10. The method according to claim 8 or 9, characterized in that, The heterotrophic culture is carried out in a constant temperature shaking incubator in a shaker, and at least meets the following requirements: temperature is 25°C and shaker speed is 120 rpm.

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