Desert algae separation and purification method for promoting growth of soil probiotics and application of desert algae separation and purification method
Separation and purification of desert algae by solid plate coating method solves the problems of difficult operation, long time and susceptibility to pollution in the existing methods, and achieves the effect of quickly obtaining high-purity desert algae species, and promotes the growth of soil probiotics.
Patent Information
- Application Number
- CN202411870795.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-03-14
AI Technical Summary
The existing desert algae separation and purification methods have problems such as difficult operation, long time, and susceptibility to pollution, making it difficult to quickly and easily obtain high-purity desert algae species.
By collecting desert soil samples containing desert algae cells, adding medium suitable for desert algae growth to resuscitation, and using solid plate coating method for isolation and purification, and finally the purified algae strain was selected through microscopic examination and bacterial detection.
It has achieved high purity of desert algae seeds in a short period of time, avoiding bacterial contamination by algae cells, and accelerating the algae seed screening process that promotes soil probiotic growth.
Smart Images
Figure CN119614372A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of separation and purification of desert algae, and in particular relates to a method for separating and purifying desert algae for promoting the growth of soil probiotics and an application thereof. Background Art
[0002] The cultivation and application of microalgae first require algae species. Algae species are first selected and purified from natural biota using certain methods to obtain pure single algae culture, which is then expanded and preserved for experimental production. Screening and purifying microalgae is an indispensable technology for scientific research. Similarly, desert microalgae are used to promote the growth of soil probiotics, thereby conducting research on soil improvement. The separation and purification of desert microalgae is the first problem that must be solved.
[0003] Desert algae is a special environment algae. The growth environment (extreme drought, large temperature difference, strong radiation, poor soil) determines the special nutritional value of cells and improves the activity of stress resistance components. It is a kind of algae that can survive in the desert. Desert algae is a pioneer organism in the desert. With the development trend of artificial large-scale cultivation of desert microalgae, it often takes several months to develop and utilize desert microalgae and screen and separate an excellent microalgae species with application value.
[0004] At present, the specific methods used for separation and purification of microalgae are: sample serial dilution separation method, micropipette separation method, water droplet separation method and solid culture medium separation method. In summary, each method has its own advantages and scope of application. The micropipette separation method is very difficult to operate and there is the possibility of bacterial contamination; the dilution separation method is not very purposeful, the separation process is long, and it is not necessarily guaranteed that the required single species can be separated; the water droplet separation method is simple to operate and is more suitable for the separation of algae species that are only contaminated by bacteria, but the separation process is long and there is the possibility of bacterial contamination. It can be seen that the above methods for separation and purification of microalgae are either very labor-intensive, the culture time is long, and they are easily contaminated in the middle; or they are difficult to operate and difficult to achieve. Although the flat solid separation method is simple to operate and theoretically easier to achieve the purpose of separating a single species, it is not known whether it is actually suitable for the separation of desert algae. Therefore, it is very necessary to invent a method suitable for rapid and simple separation and purification of Chlorella through the innovation of solid culture medium based on the growth characteristics of desert algae. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a method for separating and purifying desert algae that promotes the growth of soil probiotics and its application. The present invention collects desert soil samples containing desert algae cells, adds a culture medium suitable for the growth of desert algae for recovery, uses a preferred solid plate coating method for separation and purification, and finally selects purified algae strains through microscopic examination and fungal detection. The method of the present invention can obtain algae species with high purity in a short time, can avoid the contamination of desert algae cells by bacteria, and accelerate the screening of algae species that promote the growth of soil probiotics.
[0006] In order to achieve the above object, the present invention adopts the following technical solutions:
[0007] The present invention provides a method for separating and purifying desert algae for promoting the growth of soil probiotics, and the specific steps of the method are as follows:
[0008] A. Collect samples from the soil surface crust and below to a depth of 10 cm;
[0009] B. Dissolve the collected samples in culture medium and let them stand, and obtain algae solution after recovery;
[0010] C. After being fully diluted, the obtained algae liquid is spread on a solid culture medium for separation;
[0011] D. Take the separated single algae and drop it on a solid plate and repeatedly streak it to separate it until a single algae species is obtained;
[0012] E. Through microscopic examination and fungal detection, select the purified algae strain for expanded culture and set aside.
[0013] Furthermore, the specific process of step A is as follows: when sampling, a 1m×1m sample plot is selected, and the soil surface crust and below it to a depth of 10cm are carefully collected with a sterile shovel, and collected in a sterile sampling bottle. After each sampling, the sampling tool is wiped with 70% alcohol to prevent cross contamination.
[0014] Furthermore, the resuscitation process in step B is: taking 19-21g of sample and adding it into a sterilized 200mL conical flask containing 130mL of culture medium, placing it under the conditions of light intensity of 4500-5500Lux, light-dark cycle of 12h / 12h, temperature of 25-27°C, and culturing for 14-16 days.
[0015] Furthermore, the separation method in step C is a plate coating method, and the specific process is: after the inoculation loop is sterilized by the flame of an alcohol lamp, the algae liquid is sucked into the solid culture medium under aseptic operation conditions, and 3 to 4 first streaks are gently made on the surface of the culture medium, and then a second streak is made through the first streak area. The third and fourth streaks are made in the same way, and the streaked plate is placed on a culture rack for static culture.
[0016] Furthermore, the marking method in step C is any one of a parallel marking method, a fan-shaped marking method or other continuous marking methods.
[0017] Furthermore, the conditions for static culture in step C are: light intensity of 4500-5500 Lux, light-dark cycle of 18h:6h, and culture time of 9-11 days.
[0018] Furthermore, the separation method in step D is the same as that in step C. After the algae strain grows well, pick up a clean single algae colony, use a pipette to absorb an appropriate amount of algae liquid under sterile conditions, or use an inoculation loop / toothpick to pick up a single algae colony on the solid culture medium, and gently make 3 to 4 first streaks on the surface of the culture medium, and then make a second streak through the first streak area, and use the same method to make the third and fourth streaks; the plate after streaking is cultured until the microalgae are consistent in morphology and sterile, that is, the purification is successful.
[0019] Furthermore, the specific process of step E is: under sterile conditions, a proper amount of algae liquid to be tested is aspirated and dropped onto a glass slide to make slices, and observed under different magnification backgrounds using a differential interference microscope. If no foreign bacteria are found, it can be judged to be sterile. The sterility test observation must be carried out in a sterile room to avoid contamination of the sample by foreign microorganisms and affect the accuracy of the sterility test results. Through microscopic examination and fungal detection, the purified algae species are selected for expanded culture.
[0020] All the above experimental supplies were sterilized at high temperature (120℃, 1h) and ultraviolet (>20min) and then cooled for use.
[0021] The present invention also provides desert algae obtained by using the desert algae separation and purification method.
[0022] The present invention also provides an application of the desert algae in promoting the growth of soil probiotics.
[0023] Compared with the prior art, the present invention has the following technical effects:
[0024] The present invention collects desert soil samples containing desert algae cells, adds a culture medium suitable for the growth of desert algae for recovery, uses a preferred solid plate coating method for separation and purification, and finally selects purified algae strains through microscopic examination and fungal detection. The method of the present invention can obtain algae species with high purity in a short time, can avoid the contamination of desert algae cells by bacteria, and accelerate the screening of algae species that promote the growth of soil probiotics. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is a rendering of the desert algae obtained by the solid culture medium plate streaking separation and purification method of the present invention in Example 1 of the present invention;
[0026] Figure 2 This is a diagram showing the specific steps of the solid plate streaking separation and purification method in Example 1 of the present invention;
[0027] Figure 3 This is a diagram showing the effect of separating and purifying desert algae using the dilution separation and purification method in Comparative Example 1 of the present invention. DETAILED DESCRIPTION
[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention are within the scope of the present invention. In the examples of the present invention, the equipment, raw materials, etc. used are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.
[0029] The technical solution of the present invention is further described in detail below in conjunction with embodiments.
[0030] Example 1 Flat plate streaking method
[0031] (1) Soil sample collection: Sampling is done in the field. When sampling, select a 1m×1m sample plot and carefully collect the soil surface crust and below to a depth of 10cm with a sterile shovel and collect it in a sterile sampling bottle. After each sampling, wipe the sampling tool with 70% alcohol to prevent cross contamination.
[0032] (2) Return to the laboratory and resuscitate the soil sample. Take about 20 g of the sample and add it to a sterilized 200 mL conical flask containing 130 mL of culture medium. Place it in a room with a light intensity of 5000 Lux, a light-dark cycle of 12 h / 12 h, and a temperature of 26 °C for 7-10 days.
[0033] (3) Prepare solid culture medium.
[0034] (4) Algae species separation is carried out under sterile conditions by plate spreading method; after the inoculation loop is sterilized by the flame of an alcohol lamp, a small amount of algae liquid is drawn under sterile operation conditions and inoculated into the reserved solid culture medium. Gently make 3 to 4 first streaks on the surface of the culture medium, and then make a second streak through the first streak area. The third and fourth streaks are made in the same way. Parallel streaks, fan-shaped streaks and other continuous streaks can be used. The streaked plate is placed on a culture rack for static culture.
[0035] (5) This method is the same as the microalgae streak separation method. After the algae strain grows well, pick up a clean single algae colony. Use a pipette to absorb an appropriate amount of algae liquid or use an inoculation loop (toothpick) to pick up a single algae colony on the solid culture medium under sterile conditions; gently make 3 to 4 first streaks on the culture medium surface, and then make a second streak through the first streak area; use the same method to make the third and fourth streaks. The streaked plate is cultured under appropriate culture conditions until the microalgae are consistent in morphology and sterile, which means that the purification is successful.
[0036] (6) Under sterile conditions, an appropriate amount of the algae to be tested is dripped onto a glass slide to make a slice, and observed under a differential interference microscope at different magnification backgrounds. If no foreign bacteria are found, it can be judged as sterile. The sterility test observation must be carried out in a sterile room to avoid the sample being contaminated by foreign microorganisms and affecting the accuracy of the sterility test results. Through microscopic examination and fungal detection, the purified algae species are selected for expanded culture.
[0037] The above laboratory operations and inoculation processing operations are all carried out in an ultra-clean workbench.
[0038] The effect diagram of the desert algae obtained by the solid culture medium plate streaking separation and purification method of the present invention is as follows: Figure 1 As shown; the specific steps of the solid plate streak separation and purification method in this embodiment are shown in FIG. Figure 2 shown.
[0039] The growth of desert algae that promotes the growth of soil probiotics was observed by different separation and purification methods. Among them, the solid plate streaking method can achieve rapid growth in a short time (7-10 days). Moreover, it is easy to meet the subsequent suspension growth inoculation requirements. When it is inoculated into the solution culture medium, a higher concentration of algae liquid can be obtained.
[0040] Comparative Example 1 Dilution separation and purification method
[0041] (1) Soil sample collection: Sampling is done in the field. When sampling, select a 1m×1m sample plot and carefully collect the soil surface crust and below to a depth of 10cm with a sterile shovel and collect it in a sterile sampling bottle. After each sampling, wipe the sampling tool with 70% alcohol to prevent cross contamination.
[0042] (2) Take about 20 g of soil sample and add it into a sterilized 200 mL conical flask containing 130 mL of culture medium. Incubate it at a light intensity of 5000 Lux, a light-dark cycle of 12 h:12 h, and a temperature of 26 °C.
[0043] (3) Take 1 ml of the revived desert algae suspension and add it to a test tube containing 9 ml of sterile culture medium and mix thoroughly.
[0044] (4) Use a sterile pipette to draw 1 ml from this test tube and add it to another test tube containing 9 ml of sterile water. Mix well. Repeat this process to make sample solutions with different dilutions of 10-1, 10-2, and 10-3 (the degree of dilution depends on the cell density).
[0045] (5) Cultivate under appropriate temperature and light conditions and observe the growth of the strain regularly.
[0046] (6) Use a pipette to draw 100 μl of algae liquid and drop it into an EP tube containing 900 μl of sterile water. Continue to blow and swirl to make an algae suspension.
[0047] (7) Use a pipette to draw 100 μl of the algae suspension and drop it into a second EP tube containing 900 μl of sterile water. Dilute it 2 to 3 times according to the growth of the algae suspension (determined by the biomass or color of the algae cells).
[0048] (8) After dilution, use a pipette to draw 20-30 μl of algae liquid from the last EP tube and drop it onto the solid culture medium plate.
[0049] (9) Place a coating rod dipped in a small amount of alcohol on the flame and ignite it. After the alcohol is burned out, cool it for 8 to 10 seconds.
[0050] (10) Use a coating rod to evenly spread the algae solution on the surface of the culture medium. During the coating process, the culture dish can be rotated to ensure that the algae solution is evenly distributed. Place the coated plate on a clean rack for routine culture and observe the plate regularly.
[0051] (11) Microscopic examination and bacterial testing are performed to select sterile algae strains for expanded culture.
[0052] The above laboratory operations and inoculation processing operations are all carried out in an ultra-clean workbench.
[0053] The effect diagram of the separation and purification of desert algae by dilution separation and purification method in this comparative example is as follows Figure 3 shown.
[0054] This method is not very purposeful, the separation process is long, and it does not necessarily guarantee that the desired single species can be separated.
[0055] Comparative Example 2 Capillary Microtube Separation and Purification Method
[0056] (1) Soil sample collection: Sampling is done in the field. When sampling, select a 1m×1m sample plot and carefully collect the soil surface crust and below to a depth of 10cm with a sterile shovel and collect it in a sterile sampling bottle. After each sampling, wipe the sampling tool with 70% alcohol to prevent cross contamination;
[0057] (2) Take about 20 g of soil sample and add it to a sterilized 200 mL conical flask containing 130 mL of culture medium, and culture it under the conditions of light intensity of 5000 Lux, light-dark cycle of 12 h:12 h, and temperature of 26 °C;
[0058] (3) Prepare 50 mL centrifuge tubes in advance, sterilize them at 121°C for 30 min, and dry them for later use;
[0059] (4) Preparation and sterilization of capillary micropipette: While sampling, another person is responsible for making capillary micropipette. Use glass Pasteur pipette to clean by ultrasonic wave, soak in alcohol, dry, sterilize at 115℃ for 2h, and use inoculation loop electric heater to burn and draw. Draw the Pasteur pipette into capillary micropipette with pore diameter of about 60um. Before use, irradiate with ultraviolet light for 30min to disinfect and sterilize again;
[0060] (5) Use a sterilized 50ml centrifuge tube to sample 20mL. Take two samples. Mix the algae solution evenly before sampling. Add 20mL of ultrapure water and shake well. Let it stand for 30 minutes and discard the supernatant.
[0061] (6) The above precipitate was transferred to two sterilized 10 mL centrifuge tubes, ultrapure water was added to 9 mL, and the tubes were centrifuged at 2000 rpm for 5 min. The supernatant was discarded and the centrifugation process was repeated 3 times.
[0062] (7) Transfer the final centrifuged precipitate to two other 10 ml centrifuge tubes of American Jun, add 5 ml of ultrapure water, and mix for later use;
[0063] (8) Place a capillary micropipette into the algae solution to be separated, suck up a small amount of algae solution, blow the sucked algae solution onto a glass slide, observe under a 10x or 20x microscope, find the target algae cells to be separated in the water droplets, and use the micropipette to suck up the target algae species again.
[0064] (9) Drop the solution onto a glass slide and repeat several times until a single target algae species is obtained. Transfer the solution into a test tube containing sterilized culture medium for expansion culture.
[0065] The above laboratory operations and inoculation processing operations are all carried out in an ultra-clean workbench.
[0066] The above method has the advantage that the absorbed algae cells are single algae cells, so a monoclonal algae species can be obtained with only one separation. However, this separation method is relatively complicated to operate, and it is difficult to absorb tiny algae cells, so it is only suitable for algae with larger cells. At the same time, since it is a single algae cell culture, the cell density is very low, which can easily lead to the failure of separation and culture.
[0067] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.
Claims
1. A method for separating and purifying desert algae for promoting the growth of soil probiotics, characterized in that: The specific steps are as follows: A. Collect samples from the soil surface crust and below to a depth of 10 cm; B. Dissolve the collected samples in culture medium and let them stand, and obtain algae solution after recovery; C. After being fully diluted, the obtained algae liquid is spread on a solid culture medium for separation; D. Take the separated single algae and drop it on a solid plate and repeatedly streak it to separate it until a single algae species is obtained; E. Through microscopic examination and fungal detection, select the purified algae strain for expanded culture and set aside.
2. The method for separating and purifying desert algae according to claim 1, characterized in that: The specific process of step A is as follows: when sampling, a 1m×1m sample plot is selected, and the soil surface crust and below it to a depth of 10cm are carefully collected with a sterile shovel, and collected in a sterile sampling bottle. After each sampling, the sampling tool is wiped with 70% alcohol to prevent cross contamination.
3. The method for separating and purifying desert algae according to claim 2, characterized in that: The resuscitation process in step B is as follows: 19-21 g of sample is added to a sterilized 200 mL conical flask containing 130 mL of culture medium, and cultured for 14-16 days under conditions of a light intensity of 4500-5500 Lux, a light-dark cycle of 12 h / 12 h, and a temperature of 25-27 ° C.
4. The method for separating and purifying desert algae according to claim 3, characterized in that: The separation method in step C is a plate coating method, and the specific process is: after the inoculation loop is sterilized by the flame of an alcohol lamp, the algae liquid is sucked into the solid culture medium under aseptic operation conditions, 3 to 4 first streaks are gently made on the surface of the culture medium, and then a second streak is made through the first streaking area. The third and fourth streaks are made in the same way, and the streaked plate is placed on a culture rack for static culture.
5. The method for separating and purifying desert algae according to claim 4, characterized in that: The marking method in step C is any one of a parallel marking method, a fan-shaped marking method or other continuous marking methods.
6. The method for separating and purifying desert algae according to claim 5, characterized in that: The conditions for static culture in step C are: light intensity of 4500-5500 Lux, light-dark cycle of 18h:6h, and culture time of 9-11 days.
7. The method for separating and purifying desert algae according to claim 6, characterized in that: The separation method in step D is the same as that in step C. After the algae strain grows well, pick up a clean single algae colony, use a pipette to absorb an appropriate amount of algae liquid under sterile conditions, or use an inoculation loop / toothpick to pick up a single algae colony on the solid culture medium, gently make 3 to 4 first streaks on the culture medium plane, then make a second streak through the first streaking area, and use the same method to make the third and fourth streaks; the plate after streaking is cultured until the microalgae are consistent in morphology and sterile, that is, the purification is successful.
8. The method for separating and purifying desert algae according to claim 7, characterized in that: The specific process of step E is as follows: under sterile conditions, a proper amount of algae liquid to be tested is aspirated and dropped onto a glass slide to make slices, and observed under different magnification backgrounds using a differential interference microscope. If no foreign bacteria are found, it can be judged to be sterile. The sterility test observation must be carried out in a sterile room to avoid the sample being contaminated by foreign microorganisms and affecting the accuracy of the sterility test results. Through microscopic examination and fungal detection, the purified algae species are selected for expanded culture.
9. Desert algae obtained using the desert algae separation and purification method according to any one of claims 1 to 8.
10. Use of the desert algae according to claim 9 in promoting the growth of soil probiotics.