Nitrogen-fixing heterocyst of cyanobacteria, rapid induction method and application thereof
By adding a specific concentration of phosphorus source under nitrogen-deficient conditions and optimizing the induction method of heterocysts using a high-content cell imaging analysis system, the problem of slow induction rate of heterocysts was solved, and efficient nitrogen fixation by nitrogen-fixing cyanobacteria and preparation of bio-nitrogen fertilizer were achieved.
Patent Information
- Application Number
- CN202411581589.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2026-05-15
- Estimated Expiration
- 2044-11-07
AI Technical Summary
The induction rate of heterocysts in existing technologies is relatively slow, which limits the large-scale application of nitrogen-fixing cyanobacteria.
Quantitative analysis was performed using a high-content cell imaging analysis system. BG11-N0P2 and BG11-N0P3 culture media were prepared by adding a specific concentration of phosphorus source to nitrogen-deficient culture medium. After 12 hours of induction, the media were transferred to nitrogen-deficient and phosphorus-supplemented culture medium for further induction. The phosphorus source concentration and time were optimized to accelerate the formation of atypical cells.
It shortens the induction period of heterocysts, improves the nitrogen fixation efficiency of nitrogen-fixing cyanobacteria, and promotes the development of heterocysts by using inexpensive dipotassium hydrogen phosphate, making it suitable for the preparation of bio-nitrogen fertilizer.
Smart Images
Figure CN119685196B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of nitrogen-fixing cyanobacterial heterocysts, specifically relating to a nitrogen-fixing cyanobacterial heterocyst, a rapid induction method, and its application. Background Technology
[0002] Nitrogen-fixing cyanobacteria refer to a general term for cyanobacteria capable of fixing nitrogen. Examples include Anabaena (…). Anabaenasp. ), Nostoc ( Nostocsp. ) and filamentous algae ( Aphanizomenonsp. All three algae belong to the phylum Cyanophyta, class Cyanophyceae, order Nostocales, and family Nostocaceae. They can utilize various forms of nitrogen sources in the environment, such as ammonium salts, nitrates, and nitrogen gas. When utilizing combined nitrogen sources such as ammonium salts and nitrates, the entire hyphae consist of a single-morphological cell, which is the site of photosynthesis and is called a vegetative cell. Under conditions lacking nitrates or ammonium, about 5%-10% of the vegetative cells on the hyphae of all three algae differentiate into a type of cell with unique morphology and function, namely heterocysts. Under aerobic conditions, the vegetative cells of cyanobacteria can normally perform photosynthesis and release oxygen. Since nitrogenase is inactivated by oxygen, aerobic photosynthesis and nitrogen fixation are incompatible processes. Heterocysts differentiate into thick-walled structures to reduce oxygen permeation, thus protecting the internal nitrogenase from oxygen degradation and maintaining its activity. Nitrogenase converts nitrogen into ammonia within the heterocyst and transfers the ammonia to vegetative cells via plasmodesmata, completing the nitrogen fixation process. Heterocysts are specialized cells for nitrogen fixation in cyanobacteria, typically forming when nitrogen is scarce in the environment. The three cyanobacteria in this patent are all capable of differentiating into heterocysts for nitrogen fixation. The nitrogen fixation efficiency of these cyanobacteria is directly related to the characteristics of their heterocysts; the number, size, and nitrogenase activity of the heterocysts are all direct factors determining the nitrogen fixation efficiency. Currently, research on heterocyst induction largely focuses on optimizing the induction process by altering environmental factors such as light intensity, light-dark cycle conditions, and salt concentration. Heterocyst induction has not yet been widely adopted, possibly due to the current induction speed. With further research, future strategies aimed at optimizing nutrient conditions to improve heterocyst induction may be developed. Summary of the Invention
[0003] This invention uses a high-content cell imaging analysis system to quantify heterocysts of nitrogen-fixing cyanobacteria and, through algorithm analysis, obtains a rapid induction method for heterocysts of nitrogen-fixing cyanobacteria, providing a new option for the application of heterocysts in biotechnology.
[0004] The objective of this invention is achieved through the following technical solution:
[0005] A method for rapid induction of heterocysts from nitrogen-fixing cyanobacteria includes the following steps:
[0006] Step 1: Under light conditions, nitrogen-fixing cyanobacteria are placed in nitrogen-deficient culture medium BG11-N0 for 12 hours to induce induction.
[0007] Step 2: Take out the nitrogen-fixing cyanobacteria that have been induced in Step 1 and place them in a nitrogen-deficient and phosphorus-supplemented culture medium BG11-N0P for another 12 hours.
[0008] The concentration ratio of phosphorus source in the culture medium of Step 1 and Step 2 is 1:(2-3).
[0009] As a more preferred technical solution of the present invention: the concentration ratio of phosphorus source in the culture medium of step one and step two is 1:3.
[0010] As a preferred technical solution of the present invention: the nitrogen-deficient culture medium lacks a nitrogen source in its components compared with the BG11 culture medium.
[0011] As a more preferred technical solution of the present invention: the nitrogen-deficient and phosphorus-supplemented culture medium and the phosphorus source in the nitrogen-deficient culture medium are dipotassium hydrogen phosphate.
[0012] As a more preferred technical solution of the present invention: the concentration of dipotassium hydrogen phosphate in the nitrogen-deficient culture medium in step one is 40 mg / L, and the concentration of dipotassium hydrogen phosphate in the nitrogen-deficient phosphorus-supplemented culture medium in step two is 80-120 mg / L.
[0013] As a preferred technical solution of the present invention: the induction time for both step one and step two is 12 hours, and the total induction time is 24 hours.
[0014] As a preferred technical solution of the present invention: the nitrogen-fixing cyanobacteria are Anabaena, Nostoc, or Fibrocystidia.
[0015] As a preferred technical solution of the present invention: the light intensity is 1000 Lux, the induction temperature is 20-25℃, and the shaking culture speed is 120 r / min.
[0016] Another objective of this invention is to provide a nitrogen-fixing cyanobacterial heterocellular cell, which is prepared by the above-described rapid induction method for nitrogen-fixing cyanobacterial heterocellular cells.
[0017] Another objective of this invention is to provide the application of the aforementioned nitrogen-fixing cyanobacterial heterocysts in the preparation of bio-nitrogen fertilizers.
[0018] The beneficial effects are as follows:
[0019] This invention employs a high-content cell imaging analysis system to quantify heterocysts in nitrogen-fixing cyanobacteria and, through algorithmic analysis, derives a rapid induction method for these heterocysts. Compared to conventional induction methods (induction in nitrogen-deficient culture medium BG11-N0), this invention promotes heterocyst formation in nitrogen-fixing cyanobacteria and accelerates the nitrogen fixation process by using specific phosphorus source concentrations (the phosphorus source addition amount in BG11-N0P2 is twice that in BG11-N0, and the phosphorus source addition amount in BG11-N0P3 is three times that in BG11-N0) and culture time.
[0020] After verifying that three algal heterocysts were induced in BG11-N0 culture medium for 12 hours, they were then removed and further induced in BG11-N0P2 and BG11-N0P3 culture media for another 12 hours, the BG11-N0P3 culture medium showed the best environment for heterocyst development and also shortened the induction period.
[0021] The phosphorus source used in this invention can be dipotassium hydrogen phosphate, which is inexpensive, easy to use, and does not require large instruments or dangerous devices. Attached Figure Description
[0022] Figure 1 shows heteromorphic cell images of the three algae obtained in Examples 1, 3, 4, and 5 under high-content imaging, where a, b, and c represent the morphology of Anabaena, Nostoc, and Trichophyton in BG11, BG11-N0, BG11-N0P2, and BG11-N0P3, respectively.
[0023] Figure 2 The cell size and relative fluorescence intensity of heterocysts differentiated from three algae obtained in Examples 1, 3, 4 and 5 under different culture conditions are shown. The three algae are BG11 (sampled after Example 1), BG11-N0 (sampled after Example 3), BG11-N0P2 (sampled after Example 4) and BG11-N0P3 (sampled after Example 5).
[0024] Figure 3 shows heterocellular images of the three algae obtained in Examples 3, 6, and 7 under high-content imaging. Figures a, b, and c represent Anabaena, Nostoc, and Trichophyton in BG11-N0 (sampled after Example 3), BG11-N0P2 (sampled after Example 6), and BG11-N0P3 (sampled after Example 7), respectively.
[0025] Figure 4 The figures show the cell size and relative fluorescence intensity of heterocysts formed by the three algae obtained in Examples 3, 6 and 7 under different culture conditions. The figures are BG11-N0 (sampled after Example 3), BG11-N0P2 (sampled after Example 6) and BG11-N0P3 (sampled after Example 7).
[0026] Figure 5 shows heterocellular images of the three algae obtained in Examples 10, 8, and 9 under high-content imaging. a, b, and c represent Anabaena, Nostoc, and Trichophyton, respectively. In the figure, BG11-N0 (sampled after Example 10), BG11-N0P2 (sampled after Example 8), and BG11-N0P3 (sampled after Example 9) are also shown.
[0027] Figure 6 The cell size and relative fluorescence intensity of heterocysts differentiated from the three algae obtained in Examples 10, 8 and 9 under different culture conditions are shown.
[0028] Figure 7 Microscopic images of three algae obtained in Examples 1, 3, 4 and 5 are shown. Among them, a, b, c and d represent microscopic images of algae in culture media of BG11, BG11-N0, BG11-N0P2 and BG11-N0P3, respectively. From left to right, they are Anabaena, Nostoc, and Thymicola, BG11 (sampled after Example 1), BG11-N0 (sampled after Example 3), BG11-N0P2 (sampled after Example 4) and BG11-N0P3 (sampled after Example 5).
[0029] Figure 8 To observe the channel maps and overlay images of nitrogen-fixing cyanobacteria with differentiated heterocysts in TL10, TexasRed, and CHLA using high-content imaging (the overlay is marked in blue to represent heterocysts). Detailed Implementation
[0030] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0031] Example 1
[0032] Anabaena, Nostoc, or Trichophyton spp. were inoculated into 500mL Erlenmeyer flasks containing BG11 medium. The flasks were sealed with film and placed on a constant-temperature shaker at 120rpm, 1000Lux, and 25℃. OD values were then measured. 750 Up to 0.35. The components and contents of the BG11 culture medium are shown in Table 1 below.
[0033] Table 1
[0034]
[0035] The BG11 culture medium preparation method involves weighing each raw material according to the specified ratio, mixing and sterilizing it using conventional methods.
[0036] Example 2
[0037] Take 30 mL of each of the three algal strains cultured in BG11 medium from Example 1, centrifuge at 4000 rpm for 2 min, collect the bacterial cells, and resuspend the cells in BG11-N0 culture medium. Repeat the centrifugation and resuspension process twice. After sealing the bottle with a sealing film, place it on a constant temperature shaker and incubate at 120 rpm, 1000 Lux, 25°C for 12 hours. The BG11-N0 culture medium contains the following components and their contents, as shown in Table 2.
[0038] Table 2
[0039]
[0040] The method for preparing BG11-N0 culture medium involves weighing each raw material according to the specified ratio, mixing and sterilizing them using conventional methods.
[0041] Example 3
[0042] Take 30 mL of each of the three algal strains prepared in BG11 medium in Example 1, centrifuge at 4000 r / min for 2 min, collect the bacterial cells, add them to BG11-N0 culture medium to resuspend the bacterial cells, repeat the centrifugation and resuspension process twice, add a sealing film to the mouth of the bottle and place it on a constant temperature shaker, 120 rpm, 1000 Lux, 25℃, shake and culture for 24 hours.
[0043] The BG11-N0 culture medium preparation method involves weighing each raw material according to the specified ratio, mixing and sterilizing it using conventional methods.
[0044] Example 4
[0045] Take 30 mL of each of the three algal strains cultured in BG11 medium from Example 1, centrifuge at 4000 r / min for 2 min, collect the bacterial cells, and resuspend the cells in BG11-N0P2 culture medium. Repeat the centrifugation and resuspension process twice. After sealing the bottle with a sealing film, place it on a constant temperature shaker and incubate at 120 rpm, 1000 Lux, 25℃ for 24 hours. The BG11-N0P2 culture medium includes the following components and their contents, as shown in Table 3.
[0046] Table 3
[0047]
[0048] The BG11-N0P2 culture medium preparation method involves weighing each raw material according to the above proportions, mixing and sterilizing them using conventional methods.
[0049] Example 5
[0050] Take 30 mL of each of the three algal strains cultured in BG11 medium from Example 1, centrifuge at 4000 r / min for 2 min, collect the bacterial cells, and resuspend the cells in BG11-N0P3 culture medium. Repeat the centrifugation and resuspension process twice. After sealing the bottle with a sealing film, place it on a constant temperature shaker and incubate at 120 rpm, 1000 Lux, 25℃ for 24 hours. The BG11-N0P3 culture medium includes the following components and their contents, as shown in Table 4.
[0051] Table 4
[0052]
[0053] The BG11-N0P3 culture medium preparation method involves weighing each raw material according to the above proportions, mixing and sterilizing them using conventional methods.
[0054] Example 6
[0055] Take 30 mL of each of the three cyanobacteria strains cultured in Example 2, centrifuge at 4000 r / min for 2 min, collect the bacterial cells, and culture them in BG11-N0P2 medium for 24 h. Add a sealing film to the mouth of the bottle, and then place it on a constant temperature shaker at 120 rpm, 1000 Lux, 25 °C, and shake for culture. Take samples at 25 h.
[0056] Example 7
[0057] Take 30 mL of each of the three cyanobacteria strains cultured in Example 2, centrifuge at 4000 r / min for 2 min, collect the bacterial cells, and culture them in BG11-N0P3 medium for 24 h. Add a sealing film to the mouth of the bottle, and then place it on a constant temperature shaker at 120 rpm, 1000 Lux, 25 °C for shaking culture. Take samples at 25 h.
[0058] Example 8
[0059] Take 30 mL of each of the three cyanobacteria cultured in Example 3, centrifuge at 4000 r / min for 2 min, collect the bacterial cells, and culture them in BG11-N0P2 medium for 48 h. Add a sealing film to the mouth of the bottle, and then place it on a constant temperature shaker at 120 rpm, 1000 Lux, 25 °C, and shake for culture. Take samples at 49 h.
[0060] Example 9
[0061] Take 30 mL of each of the three cyanobacteria strains cultured in Example 3, centrifuge at 4000 r / min for 2 min, collect the bacterial cells, and culture them in BG11-N0P3 medium for 48 h. Add a sealing film to the mouth of the bottle, and then place it on a constant temperature shaker at 120 rpm, 1000 Lux, 25 °C, and shake for culture. Take samples at 49 h.
[0062] Example 10
[0063] The three cyanobacteria strains cultured for 24 hours in Example 3 were then placed on a constant temperature shaker and cultured for another 48 hours at 120 rpm, 1000 Lux, and 25°C. Samples were taken at 49 hours.
[0064] In this invention, the formulations of the culture media BG11-N0P2 and BG11-N0P3 differ from those of BG11-N0 only in that the potassium dihydrogen phosphate concentrations in BG11-N0P2 and BG11-N0P3 are 80 mg / L and 120 mg / L, respectively.
[0065] Alcian Blue was added to the three cyanobacteria obtained in the above examples, the supernatant was removed, and the hyphae were suspended in a heterocellular staining buffer. The Alcian Blue was prepared by dissolving 50 mg of the drug in 10 mL of 50% ethanol solution. The preparation method of the heterocellular staining buffer included the following steps:
[0066] A. Dissolve 0.6055g of tris(hydroxymethyl)aminomethane in 800mL of water, then add concentrated hydrochloric acid to adjust the pH to 7.8, then add water to make up to 1L, dispense and autoclave to obtain 5mM Tris-HCl.
[0067] B. 1.4612g of ethylenediaminetetraacetic acid was placed in a container of Tris-HCl, then placed in an ultrasonic instrument with an ultrasonic power of 99% and heated to 35°C until completely dissolved.
[0068] C. Then add 1.7g of BG11 medium to the solution to make a heterocytic cell staining buffer.
[0069] After observing the sample suspended in the staining buffer under a microscope, place it into a high-content cell imaging analysis system and proceed with the following steps:
[0070] 100 µL of sample was measured using a pipette and placed into a 96-well plate with a glass bottom. The plate was then placed into a high-content cell imaging analysis system. Differentiated atypical cells were labeled and analyzed and quantified by superimposing labels, and the number, size, and relative fluorescence intensity of atypical cells were analyzed.
[0071] The number of heterocysts differentiated from the three algae obtained in Examples 1, 3, 4, and 5 under different culture conditions is shown in Table 5 below. BG11 (sampled after Example 1), BG11-N0 (sampled after Example 3), BG11-N0P2 (sampled after Example 4), and BG11-N0P3 (sampled after Example 5).
[0072] Table 5
[0073]
[0074] The number of heterocysts differentiated from the three algae obtained in Examples 3, 6, and 7 under different culture conditions is shown in Table 6 below. BG11-N0 (sampled after Example 3), BG11-N0P2 (sampled after Example 6), and BG11-N0P3 (sampled after Example 7)
[0075] Table 6
[0076]
[0077] The number of heterocysts differentiated from the three algae obtained in Examples 10, 8, and 9 under different culture conditions is shown in Table 7 below.
[0078] Table 7
[0079]
[0080] Table 5 (Number of heterocysts differentiated from the three algae obtained in Examples 1, 3, 4, and 5 under different culture conditions) and Figure 2 (Cell size and relative fluorescence intensity) It can be seen that: after induction for 24 hours in BG11-N0, BG11-N0P2, and BG11-N0P3 media, respectively, the number of heterocysts induced in BG11-N0 was the highest, followed by BG11-N0P2, and the lowest in BG11-N0P3. Among them, after induction in BG11-N0, the number of heterocysts differentiated from *Anabaena globulina* was 127% higher than that of *Nostoc commune* and 911% higher than that of *Hylocereus undatus*; the size of the heterocysts differentiated from *Anabaena globulina* was 2% larger than that of *Nostoc commune* and 19% larger than that of *Hylocereus undatus*; the relative fluorescence intensity of the heterocysts differentiated from *Anabaena globulina* was 20% stronger than that of *Nostoc commune* and 30% stronger than that of *Hylocereus undatus*.
[0081] Table 6 (Number of heterocysts differentiated from the three algae obtained under different culture conditions in Examples 3, 6, and 7) and Figure 4(Cell size and relative fluorescence intensity) It can be seen that: after induction for 24 h in BG11-N0, BG11-N0P2 and BG11-N0P3 media, respectively, the number of heterocysts induced by the three algae in Example 7 was the highest compared to the traditional induction conditions (BG11-N0) in Example 3, followed by Example 3, and the lowest in Example 6. In Example 7, the number of heterocysts differentiated from Anabaena, Nostoc, and Trichophyton was 116%, 5% and 9% more than that under the conditions of Example 3, respectively; the size of the heterocysts differentiated from Anabaena, Nostoc, and Trichophyton was 11%, 11% and 13% larger than that under the conditions of Example 3, respectively; the relative fluorescence intensity of the heterocysts differentiated from Anabaena, Nostoc, and Trichophyton was 25%, 15% and 7% stronger than that under the conditions of Example 3, respectively.
[0082] As shown in Table 7 and Figure 6 The figures show the number, cell size, and relative fluorescence intensity of heterocysts differentiated from the three algae obtained in Examples 10, 8, and 9 under different culture conditions. The three algae were induced for 48 hours under BG11-N0, BG11-N0P2, and BG11-N0P3 cultures. Compared to the traditional induction condition (BG11-N0) in Example 10, the number of heterocysts induced in Example 9 was the highest for all three algae. In Example 9, the number of heterocysts differentiated from *Anabaena*, *Nostoc*, and *Tricholoma* was 73%, 19%, and 67% higher, respectively, than under the conditions in Example 10; the size of the heterocysts differentiated from *Anabaena*, *Nostoc*, and *Tricholoma* was 121%, 33%, and 13% larger, respectively, than under the conditions in Example 10; the relative fluorescence intensity of the heterocysts differentiated from *Anabaena* was 100% stronger than under the conditions in Example 10; and the relative fluorescence intensity of the heterocysts differentiated from *Nostoc* and *Tricholoma* cultured in Example 10 was 35% and 10% stronger, respectively, than under the conditions in Example 9.
[0083] In Example 7, the three algal strains induced the highest number of heterocysts under BG11-N0P3 culture conditions, and the induction time was halved compared to Example 9. Specifically, the number of heterocysts induced in Example 7 for *Anabaena*, *Nostoc*, and *Tricholoma* was 26%, 25%, and 9% higher, respectively, than in Example 9; the size of the heterocysts induced in Example 7 was 64%, 79%, and 169% larger, respectively, than in Example 9; and the relative fluorescence intensity of the heterocysts induced in Example 7 was 192%, 257%, and 267% stronger, respectively, than in Example 9. Therefore, under the condition of a 3-fold phosphorus supplementation after 12 hours of induction, the number of heterocysts differentiated from the three algal strains showed the best results.
[0084] Although heterocyst formation is primarily triggered by nitrogen deficiency, sufficient phosphorus is crucial for cellular metabolism and energy supply during cyanobacterial growth and development. The sufficiency of phosphorus sources affects the overall growth status of cyanobacteria, thus indirectly influencing heterocyst development. In nitrogen-deficient but phosphorus-sufficient environments (high phosphorus, low nitrogen), more heterocyst differentiation is induced to maintain the population's nitrogen-fixing capacity. Sufficient phosphorus sources can indirectly affect heterocyst formation by regulating the growth rate, metabolic state, and nitrogen demand of cyanobacteria.
[0085] The three cyanobacteria in this invention are all capable of differentiating into heterocysts for nitrogen fixation. The nitrogen fixation efficiency of these cyanobacteria is directly related to the characteristics of their heterocysts; the number, size, and nitrogenase activity of the heterocysts are all direct factors determining the nitrogen fixation efficiency. Conventional induction methods involve culturing in a nitrogen-deficient culture medium. However, in this invention, phosphorus is supplemented in the nitrogen-deficient culture medium. This supplements the phosphorus required by the cyanobacteria, promoting cell metabolism and energy supply, and inducing cell differentiation. Furthermore, phosphorus supplementation enhances the degree of nitrogen deficiency, strengthening the induction environment for heterocysts.
[0086] In summary, the above embodiments mainly describe a preferred method of the present invention, including the basic operating steps, main features, and advantages of the present invention. Those skilled in the art should have a certain understanding of these methods. The present invention is not limited to the above embodiments. While the present invention has been described in detail above with general descriptions and specific implementation schemes, modifications or improvements can be made based on it, which are obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for rapid induction of heterocysts in nitrogen-fixing cyanobacteria, characterized in that: Step 1: Under light conditions, nitrogen-fixing cyanobacteria are placed in a nitrogen-deficient culture medium for 12 hours to induce induction. Step 2: Take out the nitrogen-fixing cyanobacteria induced in Step 1 and place them in a nitrogen-deficient and phosphorus-supplemented culture medium for another 12 hours; the concentration ratio of phosphorus source in the culture medium of Step 1 and Step 2 is 1:
3. The phosphorus source in the nitrogen-deficient culture medium in step one is dipotassium hydrogen phosphate; the concentration of dipotassium hydrogen phosphate is 40 mg / L; the nitrogen-deficient culture medium lacks a nitrogen source in its components compared to BG11 culture medium. The phosphorus source in the nitrogen-deficient phosphorus replenishment culture medium in step two is dipotassium hydrogen phosphate; the concentration of dipotassium hydrogen phosphate is 120 mg / L. The nitrogen-fixing cyanobacteria mentioned are Anabaena and Nostoc. The light intensity was 1000 Lux, the induction temperature was 20-25℃, and the shaking culture speed was 120 r / min.