CN114107140A - Method for in-situ sterile enrichment culture of synechococcus - Google Patents
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Abstract
The invention belongs to the technical field of aseptic culture of marine pico-type blue algae, and particularly relates to a method for in-situ aseptic enrichment culture of synechococcus. Inoculating synechococcus into an SNAX culture medium, culturing to a late logarithmic phase under the conditions of 24-26 ℃ and 1600-2400 lx, culturing the cultured synechococcus in the dark for 24-36 h to enable the synechococcus to be always in a split and lag phase, adding a mixed antibiotic consisting of streptomycin, gentamicin sulfate and neomycin sulfate into the culture medium, continuously culturing in the dark for 6-12 h to complete the life history, and culturing the growing synechococcus in the manner until the bacteria in the culture medium and the synechococcus cells are removed, thereby realizing the sterile culture of the in-situ synechococcus. The mixed antibiotic treatment method adopted by the invention successfully obtains the Synechococcus sterile enriched sample from the original position.
Description
Technical Field
The invention belongs to the technical field of aseptic culture of marine pico-type blue algae, and particularly relates to a method for in-situ aseptic enrichment culture of synechococcus.
Background
Synechococcus is a pico-type blue algae which is located throughout the world and has an important role in the geochemical cycle of marine organisms. The organic matter produced by synechococcus accounts for 16.7% of net primary production (net primary production) of ocean in the world, and the light-capturing antenna of the photosynthetic system contains abundant phycocyanin, so that the light energy can be utilized for fixationCarbon, and can fix nitrogen, and when the nitrogen source is lacked in the environment, phycocyanin can degrade by itself to release the nitrogen source. The highest abundance of synechococcus in the sea can reach 106one/mL, which is not only abundant, in terms of genetic development, there are at least 20 genetic branches, some of which exhibit a clear ecological preference, occupying different niches; the synechococcus cells contain rich phycocyanin and have important medical value, in addition, the combination of oxidative photosynthesis, carbon fixation and metabolic pathways enables the synechococcus to continuously and directly produce biofuel, bioplastic, sugar and chemical raw materials from carbon dioxide, light and water, so the synechococcus can be applied to the production of biological products through a genetic engineering technology, and the synechococcus becomes a large biological resource bank to be developed and has certain economic value, so the research on the response of the physiological and ecological characteristics of the synechococcus to environmental factors, the development and utilization of the biological resources of the synechococcus and the deep research on the value of the synechococcus in the biological engineering technology have important significance. The aseptic culture is indispensable in the physiological and ecological research and bioengineering application of the marine microalgae, and is a prerequisite for deeply researching and developing and utilizing the microalgae resources. Previous studies found that there were always associated heterotrophic bacteria around synechococcus, whether in situ or under culture conditions, and that it was difficult to separate them; meanwhile, the synechococcus is a prokaryotic cell, the synechococcus cell is easy to kill by antibiotic treatment, and the difficulty in obtaining sterile synechococcus is increased, so that the previous research on the physiological ecology and the biological engineering application of the synechococcus is actually based on a mixture of the synechococcus and heterotrophic bacteria, on one hand, the physiological ecology and the interaction mechanism between the synechococcus cannot be accurately researched due to the existence of the heterotrophic bacteria, on the other hand, a high-yield synechococcus biological product cannot be obtained, and the effect of the synechococcus biological product can be deeply researched. The existence of the above problems makes research work and resource development difficult.
The conventional method for obtaining the sterile microalgae mainly comprises physical methods such as filtering washing, centrifugal elution, ultraviolet killing and the like, the methods can only be applied to the sterilization treatment in a primary stage, and meanwhile, the ultraviolet irradiation can only temporarily delay the growth of bacteria, but can kill algae cells at the same time, so that certain defects exist. Antibiotic sterilization is also used, but due to the specificity of the antibiotic's antibacterial function, a single antibiotic cannot kill all bacteria in the intercalary environment, and sterile strains of algae cannot be obtained. Therefore, according to the sensitivity difference of the algae cells and bacteria to antibiotics, the bacteria coexisting with or polluted by the algae in the intercellular environment can be removed by using a method of mixing the antibiotics. In addition, as microalgae cells need to rely on light energy for biological carbon fixation to realize division and proliferation of the cells, the cell proliferation is in a stasis state under the condition of keeping out of the sun, and bacteria can still continuously divide and proliferate, antibiotics which obstruct the cell wall formation and act on the cell division stage can be selected for sterilization treatment under the condition, and the damage of the antibiotics to the algae cells can be reduced.
Therefore, the previous research on how to realize 'degerming and no algae removal' of the synechococcus to obtain the sterile synechococcus is to obtain the sterile synechococcus through a continuous dilution method, the method is not ideal in effect, a large amount of associated bacteria still exist, the culture time is required to be several months, and the experiment period is long. In addition, as the interstellar bacteria have tiny body types, simple structure and strong environment adaptability and can quickly proliferate, the bacteria removal is more difficult; the synechococcus belongs to prokaryotic micro-blue algae, has great similarity with bacteria, and is easy to damage the synechococcus cells by using antibiotics to treat algae liquid, so that the degerming is difficult. Therefore, it is urgently needed to find a method which has good effect, low cost, strong operability and no harm and can obtain sterile pure synechococcus cells by enrichment culture.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for in-situ sterile enrichment culture of synechococcus.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for in-situ aseptic enrichment culture of synechococcus includes inoculating synechococcus into SNAX culture medium, culturing at 24-26 deg.C and 1600-2400 lx to late logarithmic phase, culturing the cultured synechococcus in dark for 24-36 hr to make the synechococcus always in the phase of stagnation, adding mixed antibiotics of streptomycin, gentamicin sulfate and neomycin sulfate, culturing in dark for 6-12 hr to complete its life history, and repeating culture until the culture medium and the bacteria in synechococcus are removed.
And centrifuging the algae solution which is cultured in the dark after repeated culture and sterilization, collecting precipitates, washing the precipitates by using sterilized natural seawater, resuspending the precipitates by using the sterilized natural seawater, and then inoculating the precipitates into an SNAX culture medium to obtain the enrichment culture sterile synechococcus solution.
The repeated culture and sterilization are that the algae liquid which is processed by antibiotics and is cultured in the dark is inoculated into an SNAX culture medium according to the volume ratio of 1:10, then is cultured to the late logarithmic phase under the conditions of 24-26 ℃ and 1600-.
The synechococcus is S5.1 Clade VIII synechococcus.
Adding 2.90-3.10 mL of mixed antibiotic solution into every 100 mL of algae solution; wherein, the streptomycin solution, the gentamicin sulfate solution and the neomycin sulfate solution are mixed according to the volume ratio of 1:1: 1.
The streptomycin, the gentamicin sulfate and the neomycin sulfate are respectively dissolved by sterilized ultrapure water to prepare antibiotic working solution with the concentration of 49-51 g/L, 49-51 g/L and 49-51 g/L in sequence.
The invention has the advantages that:
the invention realizes 'degerming and no algae removal' by means of a mixed antibiotic treatment method in the fission stagnation stage of the synechococcus, thereby obtaining the sterile synechococcus, and the obtained sterile synechococcus can be used for physiological and ecological research, particularly the interaction of bacteria and algae, and processing and producing related bioengineering products.
The method eliminates the interference of other microorganisms, and compared with a pico-type algae-bacterium mixture, the method not only can objectively and truly reflect the response of the synechococcus to the environmental stress and influence on heterotrophic bacteria closely related to the synechococcus and community structures thereof, but also can further deeply carry out the research on the physiological and ecological characteristics of the synechococcus, the interaction between bacteria and algae and the mechanism thereof; in addition, a reference method can be provided for improving the yield of the phycocyanin of the synechococcus.
Drawings
FIG. 1 is a plot of the results of high throughput sequencing colony composition for in situ samples and enriched samples of the present invention, wherein the phylogenetic tree is a plot of the OTU representative sequences in situ samples against known developmental branches of Synechococcus using maximum likelihood.
FIG. 2 is a graph showing the bacteriostatic effect of various antibiotics provided in example 1 of the present invention.
FIG. 3 is a comparison of the present invention in example 2 with the enriched Synechococcus fluid without bacteria removal, wherein the left column shows the absence of antibiotics and the right column shows the addition of antibiotics.
FIG. 4 is a comparison graph of the present invention in example 3 with the enriched Synechococcus fluid without bacteria removal, wherein the left graph shows that no antibiotics are added, and the right graph shows that antibiotics are added.
FIG. 5 is a microscopic picture of the sterilized enriched Synechococcus fluid treated in example 3.
FIG. 6 is a comparison between comparative example 1 and the non-sterilized enriched Synechococcus fluid, wherein the left figure shows the absence of antibiotics and the right figure shows the addition of antibiotics.
FIG. 7 is a comparison between comparative example 2 and the non-sterilized enriched Synechococcus fluid, wherein the left figure shows the absence of antibiotic and the right figure shows the addition of antibiotic.
FIG. 8 is a graph showing absorbance spectra measured in an ultraviolet spectrophotometer of example 3 and comparative example 2, which were inoculated into a fresh medium after completion of sterilization according to the present invention and grown for 20 days.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the research field of synechococcus at home and abroad, the algal species are mostly mixtures of synechococcus and bacteria, the synechococcus generally exists in the global oceans, the phycocyanin content is high, the synechococcus becomes a biological resource to be developed, and the development process is hindered by the existence of the bacteria-algae mixtures. Common methods for removing synechococcus bacteria at home and abroad comprise filtration, ultraviolet radiation, antibiotic application, continuous dilution and the like, and the methods have undesirable effects and can cause great damage to the synechococcus itself in the operation process. The method of the invention uses the mixed antibiotics to sterilize when the synechococcus is in the division and stagnation stage and the bacteria are still in the division and proliferation stage, the method is feasible and has strong operability, and the synechococcus cells can not be damaged. The acquisition of the degerming synechococcus provides convenience for the physiological and ecological research and the development of biological resources of the synechococcus in the future, and has long-term ecological and bioengineering significance.
The following examples to enrich aseptically cultured Synechococcus as Clade VIII of S5.1 are known algal strains that exhibit the apparent characteristics of pigment PT3 type (Six, C., Thomas, J. C., Garczarek, L. et al. division and evolution of bacteriophages in marine)SynechococcusA synthetic genetics study, Genome Biol, 2007, 8(12): R259), which is available from the yellow sea in situ seawater (see FIG. 1). The strain is cultured in a light incubator at 24-26 ℃ under the conditions of 1600-.
Example 1
1) Culturing the algal strains: the strain is cultured in SNAX culture medium at 25 deg.C under illumination intensity of 2000 lx and light-dark ratio of 12 h: 12 h.
2) Preparing antibiotics: six antibiotics, namely Kanamycin Sulfate (Km), Ampicillin (Ampicilin, Amp), Cefotaxime (CTX), Streptomycin Solution (STR), Gentamicin Sulfate (Gm) and Neomycin Sulfate (N), are weighed respectively and prepared into an antibiotic Solution with the mass concentration of 10 g/L by sterilizing ultrapure water.
3) Antibiotic bacteriostasis: coating 100 μ L of Synechococcus liquid culture medium cultured to logarithmic phase on 2216E solid culture medium; soaking a filter paper sheet with the diameter of 5.5 mm in the above antibiotic solutions, and attaching 4 filter paper sheets to each plate (one plate uses only one antibiotic); culturing at 25 deg.C for 3 days, and measuring the size of zone of inhibition. The results of the zone of inhibition are shown in table 1, and the inhibitory effect is shown in fig. 2.
The method for testing the antibacterial effect comprises the following steps: coating plates, sucking 100 mu L of synechococcus culture solution growing to logarithmic phase, coating on 2216E solid culture medium, sticking 4 filter paper sheets soaked with the same antibiotic on each plate, sealing, marking, culturing for 3 days at 25 ℃, and measuring the size of the inhibition zone.
It can be seen from FIG. 2 that after 3 days of culture at 25 ℃, the filter paper sheets impregnated with the three antibiotics Km, Amp and CTX had bacterial colonies around them and had no significant zone of inhibition, while the filter paper sheets impregnated with the three antibiotics STR, Gm and N had almost no bacterial colonies around them and had significant zone of inhibition.
TABLE 1
Example 2
Further verification was made using the conclusions of example 1 above:
1) culturing the algal strains: the strain is grown in SNAX culture medium, and cultured to logarithmic phase in illumination incubator with 25 deg.C, illumination intensity of 2000 lx, and light-dark ratio of 12 h: 12 h.
2) Preparing antibiotics: and respectively sterilizing STR, Gm and N with ultrapure water to prepare antibiotic solutions with mass concentrations of 50 g/L.
3) Sterile enrichment culture: culturing the synechococcus liquid culture solution cultured to logarithmic growth phase at 25 ℃ in the dark for 30 h to enable the synechococcus to be in a division stagnation phase; after the light-resistant culture is finished, taking three parts of the enriched synechococcus liquid, adding one antibiotic solution into each part of the algae liquid according to the dose of adding 1 mL of the antibiotic solution into each 100 mL of the enriched synechococcus liquid, and continuing the light-resistant culture for 12 hours; after the light-resistant culture is finished, centrifugally washing the culture solution for 4 times by using sterilized natural seawater, then resuspending the culture solution by using the sterilized natural seawater, inoculating the culture solution into a sterilized SNAX culture medium according to the volume ratio of 1:10, culturing the culture solution in an illumination incubator at the temperature of 25 ℃, the illumination intensity of 2000 lx and the light-dark ratio of 12 h: 12 h, and then checking the sterilization effect of the culture solution by using a plate coating method;
meanwhile, Synechococcus fluid which was not treated with antibiotics was used as a control, and then it was examined for sterilization effect by the method of coating a plate (see FIG. 3).
The method for testing the sterilization effect comprises the following steps: coating a flat plate, sucking 20 mu L of synechococcus culture solution which is centrifuged to remove antibiotics, coating the synechococcus culture solution on a 2216E solid culture medium, sealing, marking, culturing in an incubator at 25 ℃, observing whether bacterial colonies are generated after 48 h, and taking the synechococcus culture solution which is not subjected to sterilization operation as a control.
As can be seen from FIG. 3, after culturing at 25 ℃ for 48 h, countable bacterial colonies spread on the Synechococcus culture medium plate without sterilization, and countable bacterial colonies are generated on the Synechococcus culture medium plate with different antibiotics, but the effect is to be improved.
Example 3
1) Culturing the algal strains: the strain is grown in SNAX culture medium, and cultured to logarithmic phase in illumination incubator with 25 deg.C, illumination intensity of 2000 lx, and light-dark ratio of 12 h: 12 h.
2) Preparing antibiotics: dissolving STR, Gm and N in sterilized ultrapure water to prepare antibiotic solutions with mass concentrations of 50 g/L, 50 g/L and 50 g/L respectively, and mixing the antibiotic solutions according to a volume ratio of 1:1:1 to obtain the mixed antibiotic working solution.
3) Sterile enrichment culture: culturing the synechococcus liquid culture solution cultured to logarithmic growth phase at 25 ℃ in the dark for 30 h to enable the synechococcus to be in a division stagnation phase; after the light-resistant culture is finished, adding 3 mL of antibiotic working solution into every 100 mL of synechococcus solution, and continuing the light-resistant culture for 12 h; after the light-resistant culture is finished, centrifugally washing the culture solution for 4 times by using sterilized natural seawater, then resuspending the culture solution by using the sterilized natural seawater, inoculating the culture solution into a sterilized SNAX culture medium according to the volume ratio of 1:10, culturing the culture solution in an illumination incubator with the illumination intensity of 2000 lx and the light-dark ratio of 12 h: 12 h, then carrying out the test of the sterilization effect by using a plate coating method, and testing whether the synechococcus cells are greatly damaged by using the antibiotic treatment by using a microscope microscopy method (see figure 5);
meanwhile, Synechococcus fluid which was not treated with antibiotics was used as a control, and then it was examined for sterilization effect by the method of coating a plate (see FIG. 4).
The method for testing the sterilization effect comprises the following steps: coating a flat plate, sucking 20 mu L of synechococcus culture solution which is centrifuged to remove antibiotics, coating the synechococcus culture solution on a 2216E solid culture medium, sealing, marking, culturing in an incubator at 25 ℃, observing whether bacterial colonies are generated after 48 h, and taking the synechococcus culture solution which is not subjected to sterilization operation as a control.
As can be seen from FIG. 4, after culturing at 25 ℃ for 48 hours, the colonies of bacteria were not counted on the Synechococcus culture plates without the sterilization treatment, and no colonies were generated on the Synechococcus culture plates with the sterilization treatment.
As can be seen from FIG. 5, the coccoid synechococcus cells are distributed throughout the field of view of the microscope, the growth of the synechococcus is good, and the synechococcus is not greatly damaged by the antibiotic treatment.
Comparative example 1
1) Culturing the algal strains: the strain is cultured in SNAX culture medium at 25 deg.C under illumination intensity of 2000 lx and light-dark ratio of 12 h: 12 h, and cultured to logarithmic phase.
2) Preparing antibiotics: STR, Gm and N are prepared into antibiotic solutions with mass concentrations of 50 g/L, 10 g/L and 50 g/L respectively, and the three antibiotic solutions are mixed according to the volume ratio of 1:1:1 to serve as a mixed antibiotic working solution.
3) Sterile enrichment culture: keeping the synechococcus liquid culture solution cultured to logarithmic growth phase away from light for 30 h to enable the synechococcus to be in a splitting stagnation phase; adding 3 mL of antibiotic working solution into every 100 mL of enriched synechococcus solution after the light-proof culture is finished, and continuing the light-proof culture for 12 h; after the light-resistant culture is finished, centrifugally washing the culture solution for 4 times by using sterilized natural seawater, then resuspending the culture solution by using the sterilized natural seawater, inoculating the culture solution into a sterilized SNAX culture medium according to the volume ratio of 1:10, culturing the culture solution in an illumination incubator at the temperature of 25 ℃, the illumination intensity of 2000 lx and the light-dark ratio of 12 h: 12 h, and then checking the sterilization effect of the culture solution by using a plate coating method;
meanwhile, Synechococcus fluid which was not treated with antibiotics was used as a control, and then it was examined for sterilization effect by the method of coating a plate (see FIG. 6).
The method for testing the sterilization effect comprises the following steps: coating a flat plate, sucking 20 mu L of synechococcus culture solution which is centrifuged to remove antibiotics, coating the synechococcus culture solution on a 2216E solid culture medium, sealing, marking, culturing in an incubator at 25 ℃, observing whether bacterial colonies are generated after 48 h, and taking the synechococcus culture solution which is not subjected to sterilization operation as a control.
As can be seen from FIG. 6, after culturing at 25 ℃ for 48 hours, bacterial colonies were distributed over the Synechococcus culture plates that were not subjected to the sterilization treatment, and bacterial colonies were generated on the Synechococcus culture plates that were subjected to the sterilization treatment.
Comparative example 2
1) Culturing the algal strains: the strain is cultured in SNAX culture medium at 25 deg.C under illumination intensity of 2000 lx and light-dark ratio of 12 h: 12 h, and cultured to the decline stage.
2) Preparing antibiotics: STR, Gm and N are respectively prepared into antibiotic solutions with mass concentrations of 50 g/L, 50 g/L and 50 g/L, and the three antibiotic solutions are mixed according to a volume ratio of 1:1:1 to serve as a mixed antibiotic working solution.
3) Sterile enrichment culture: culturing the synechococcus liquid culture solution cultured to the decline stage according to the step 1) for 30 hours in the dark; adding 3 mL of antibiotic working solution into every 100 mL of enriched synechococcus solution after the light-proof culture is finished, and continuing the light-proof culture for 12 h; after the light-resistant culture is finished, centrifugally washing the culture solution for 4 times by using sterilized natural seawater, then resuspending the culture solution by using the sterilized natural seawater, inoculating the culture solution into a sterilized SNAX culture medium according to the volume ratio of 1:10, culturing the culture solution in an illumination incubator at the temperature of 25 ℃, the illumination intensity of 2000 lx and the light-dark ratio of 12 h: 12 h, and then checking the sterilization effect of the culture solution by using a plate coating method;
meanwhile, Synechococcus fluid which was not treated with antibiotics was used as a control, and then it was examined for sterilization effect by the method of coating a plate (see FIG. 7).
The method for testing the sterilization effect comprises the following steps: coating a flat plate, sucking 20 mu L of synechococcus culture solution which is centrifuged to remove antibiotics, coating the synechococcus culture solution on a 2216E solid culture medium, sealing, marking, culturing in an incubator at 25 ℃, observing whether bacterial colonies are generated after 48 h, and taking the synechococcus culture solution which is not subjected to sterilization operation as a control.
As can be seen from FIG. 7, after culturing at 25 ℃ for 48 hours, bacterial colonies spread over the Synechococcus culture plates that were not subjected to the sterilization operation, and a plurality of bacterial colonies were produced on the Synechococcus culture plates that were subjected to the sterilization operation;
inoculation into new medium after sterilization for 20 days example 3 showed good growth and also the pigmentary character of synechococcus; the degerming algae liquid of the comparative example 2 is always white or even semitransparent, and does not show the normal growth state and pigment characteristics of synechococcus; as can be seen from the uv-vis scan of fig. 8, example 3 has a higher absorbance value than comparative example 2.
The technical effects of the present invention will be described in detail with reference to experiments.
The method can be used for further researching the physiological and ecological functions of the synechococcus and developing biological resources of the synechococcus. For example, the study of the material circulation in the interaction of synechococcus and related heterotrophic bacteria can provide theoretical support for the carbon and nitrogen circulation of the ocean; the synechococcus cells contain rich phycocyanin, have high medical value and are a biological resource to be developed, and the synechococcus sterile enrichment culture method provided by the invention can provide method reference for the application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A method for in-situ sterile enrichment culture of synechococcus is characterized by comprising the following steps: inoculating synechococcus into an SNAX culture medium, culturing to a late logarithmic phase under the conditions of 24-26 ℃ and 1600-2400 lx, culturing the cultured synechococcus in a dark place for 24-36 h to enable the synechococcus to be always in a split stagnation phase, adding a mixed antibiotic consisting of streptomycin, gentamicin sulfate and neomycin sulfate into the culture solution, continuously culturing for 6-12 h in the dark place to enable the synechococcus to complete the life history, and then repeatedly culturing according to the method until the culture solution and the bacteria in the synechococcus cells are removed, thereby realizing the sterile culture of the in-situ synechococcus.
2. The method of claim 1 for the in situ sterile enrichment culture of synechococcus, wherein: and centrifuging the algae solution which is cultured in the dark after repeated culture and sterilization, collecting precipitates, washing the precipitates by using sterilized natural seawater, resuspending the precipitates by using the sterilized natural seawater, and then inoculating the precipitates into an SNAX culture medium to obtain the enrichment culture sterile synechococcus solution.
3. The method of claim 1 for the in situ sterile enrichment culture of synechococcus, wherein: the repeated culture and sterilization are that the algae liquid which is processed by the antibiotics and is cultured in the dark is inoculated into the SNAX culture medium according to the volume ratio of 1:10, then is cultured to the late logarithmic phase under the conditions of 24-26 ℃ and 1600-.
4. The method of claim 1 for the in situ sterile enrichment culture of synechococcus, wherein: the synechococcus is S5.1 Clade VIII synechococcus.
5. The method of claim 1 for the in situ sterile enrichment culture of synechococcus, wherein: adding 2.90-3.10 mL of mixed antibiotic solution into every 100 mL of algae solution; wherein, the streptomycin solution, the gentamicin sulfate solution and the neomycin sulfate solution are mixed according to the volume ratio of 1:1: 1.
6. The method of claim 5, wherein the method comprises the steps of: the streptomycin, the gentamicin sulfate and the neomycin sulfate are respectively dissolved by sterilized ultrapure water to prepare antibiotic working solution with the concentration of 49-51 g/L, 49-51 g/L and 49-51 g/L in sequence.
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