CN114107061B - Seawater microalgae culture medium - Google Patents
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Abstract
The application discloses a seawater microalgae culture medium and application, wherein each liter of culture medium comprises 60-120mg of nitrogen salt, 8-12mg of phosphorus salt, 2.5-3.5mg of ferric salt and Na 2 EDTA 10-12mg, sodium 2-phosphoglycerate 0.01-0.1mg, sodium formate 0.5-1mg, sodium silicate 0-10mg, sodium diacetate 0.5-1g, and sea water the rest, wherein the analogue of algae anabolic intermediate product 2-phosphoglycerate is introduced into the culture medium in breakthrough manner, after a large number of experiments, the sodium formate and sodium diacetate which are rare in the field are determined to be matched with the sodium 2-phosphoglycerate, and the nitrogen salt, the phosphate, the ferric salt and the Na are quantified 2 EDTA and sodium silicate are matched to supplement nutrition required by culture and promote growth, and the formula is obtained according to experiments, so that the growth speed of microalgae is greatly improved.
Description
Technical Field
The application relates to the technical field of algae seed culture, in particular to a seawater microalgae culture medium.
Background
Microalgae, mostly planktonic unicellular algae with a size not exceeding 10 microns. The feed has no cell wall, is rich in unsaturated fatty acid, has strong ecological adaptability and high growth speed, is suitable for large-scale propagation, is considered as ideal feed for artificial breeding of marine animals, and is used as high-quality open feed for rare aquatic products such as mussels, oysters, arca subcrenata, prawns and the like.
In the process of culturing microalgae by adopting a traditional microalgae culture medium, the following problems exist: when seed culture is performed in the initial stage, algae tend to grow slowly, resulting in a long activation time required to culture the algae. The traditional culture base is adopted to achieve the aim of improving the culture speed of microalgae or other purposes by optimizing the formula composition of nitrogen salt, phosphate, ferric salt and the like, for example, the document of application publication No. CN103232939A, named as a marine microalgae expansion culture solution, discloses that urea, potassium dihydrogen phosphate and sodium silicate are mixed with fish feed soaking solution, the highest propagation density and the maintenance time of the platform period are far higher than those of the conventional culture nutrient solution, but the propagation speed of the marine microalgae is general. Many optimization cases in this direction show less influence on the algae cultivation, the concentration growth of algae is not high enough, and enough time is needed to provide enough first-order seeds, which may affect the production rhythm.
For this, I intend to introduce new components into the culture medium and optimize the matched components to achieve the aim of remarkably promoting the growth of microalgae.
Disclosure of Invention
In order to solve at least one technical defect, the application provides the following technical scheme:
the application discloses a seawater microalgae culture medium, which comprises 60-120mg of nitrogen salt, 8-12mg of phosphorus salt, 2.5-3.5mg of ferric salt and Na per liter of culture medium 2 10-12mg of EDTA, 0.01-0.1mg of sodium 2-phosphoglycerate, 0.5-1mg of sodium formate, 0-10mg of sodium silicate, 0.5-1g of sodium diacetate and the balance of seawater.
In the scheme, analogue sodium 2-phosphoglycerate of algae anabolic intermediate is introduced into the culture medium in breakthrough manner, and a large number of experiments prove that sodium formate, sodium diacetate and sodium 2-phosphoglycerate which are rare in the field are matched, and quantitative nitrogen salt, phosphate, ferric salt and Na are adopted 2 EDTA and sodium silicate are matched to supplement nutrition required by culture and promote growth. According to the experiment, the growth speed of the microalgae is greatly improved under the formula.
In addition, sodium silicate is required to be contained in the culture medium in the culture of, for example, golden algae and diatom, and sodium silicate may not be contained in other suitable marine chlorella besides golden algae and diatom.
The components acting in microalgae culture are presumed as follows: sodium 2-phosphoglycerate as an analogue of an intermediate in anabolism of algae may contribute to carbon assimilation in photosynthesis, sodium formate and sodium diacetate at certain concentrations may have an effect of inhibiting the amplification of bacteria and fungi in algae liquid, and in addition, sodium diacetate may also act as a carbon source during the growth of algae.
Further, the nitrogen salt comprises one or more of nitrate, ammonium salt or urea, wherein the nitrate, ammonium salt or urea is the common nitrogen salt in the culture medium, and can be singly used or two or three of the nitrogen salts are used in a matched way.
Preferred nitrates include one or more of sodium nitrate, potassium nitrate, calcium nitrate, and preferred ammonium salts include one or more of ammonium nitrate, ammonium chloride, ammonium sulfate.
Further, the ferric salt comprises one or more of ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, lin Mengsuan iron and ferric ammonium citrate, which are common ferric salt components in the culture medium, and preferably ferric ammonium citrate is adopted.
Further, the phosphate salt includes one or more of sodium salt and potassium salt containing monohydrogen phosphate or dihydrogen phosphate, which are common phosphate components in the culture medium, preferably potassium dihydrogen phosphate, and other phosphates can be selected according to the needs.
Further, each liter of the culture medium comprises potassium nitrate or NaNO 3 60-120mg，KH 2 PO 4 8-12mg, ferric ammonium citrate 2.5-3.5mg, na 2 10-12mg of EDTA, 0.01-0.1mg of sodium 2-phosphoglycerate, 0.5-1mg of sodium formate, 0-10mg of sodium silicate, 0.5-1g of sodium diacetate and the balance of seawater.
The application discloses application of the seawater microalgae culture medium in seawater microalgae culture, and the microalgae is inoculated into the microalgae culture medium for culture;
or when the primary seeds of the microalgae are activated, a small amount of preserved seeds are directly inoculated into a culture medium for culture;
or when microalgae are subjected to primary seed culture, directly inoculating the activated seeds into a culture medium for culture.
When the culture medium is used, microalgae can be directly inoculated according to the needs, or primary seeds and primary seeds are inoculated and cultured, and the culture medium has no special requirements on culture conditions and can be used according to the conventional method.
Further, the seawater microalgae comprise chlorella seawater, microcystis, golden seawater algae, diatom or rhodococcus.
Further, the diatom comprises Chaetoceros, haematococcus, diamond-shaped algae or Eggshell algae.
Compared with the prior art, the application has the beneficial effects that:
1. the application uses 2-sodium phosphoglycerate, sodium formate, sodium diacetate and sodium silicate to match with ferric salt, phosphate, nitrogen salt and Na in sea water 2 EDTA is mixed to form a culture medium, so that the growth speed of the seawater microalgae can be greatly improved, and especially the growth and propagation speed of the seawater microalgae is 10 times higher than that of a control group when the seawater Chlorella pyrenoidosa is subjected to 1-stage culture.
Drawings
FIG. 1 is a graph showing the growth rate trend of dinoflagellates 3011, such as the ball of example 1, and a control group;
FIG. 2 is a graph showing the growth rate trend of the seaweed on the control group and the seaweed on the control group in example 2;
FIG. 3 is a graph showing growth rates of dinoflagellates 3011, such as the ball of example 3, and a control group;
FIG. 4 is a graph of growth rates of the seaweed on the control group versus the seaweed on example 4;
FIG. 5 is a graph showing growth rates of Chlorella pyrenoidosa of example 5 versus a control group;
wherein the horizontal axis in the graph represents growth time in days d; the vertical axis represents the concentration of algal cells in units of 10 4 cells/mL。
Detailed Description
The application will be further described with reference to the drawings and the specific examples.
1. Culturing
Example 1
In the embodiment, the cultivation test is carried out by using the dinoflagellate 3011 seeds such as the globes and the like, and the specific steps are as follows:
1. according to the content of NaNO in each liter of culture medium 3 60mg，NaH 2 PO 4 8mg, ferric ammonium citrate 2.5mg, na 2 EDTA10mg of sodium 2-phosphoglycerate 0.1mg, 0.5mg of sodium formate, 5mg of sodium silicate, and the balance of seawater which is used for preparing a culture medium and sterilizing the culture medium by a sterilizing pot at 115 ℃ for 15 min.
2. The golden algae 3011, which was kept under low light for about 2 months, was inoculated into a 250mL Erlenmeyer flask (150 mL medium) and the above-described cooled medium and f/2+Si medium, and the culture was performed under the same conditions with the f/2+Si medium, and this operation was performed on an ultra clean bench as a control group.
3. Placing the inoculated algae into an illumination incubator for static culture, shaking three times a day for culture, and illuminating for 10 hours a day with illumination intensity of about 25 mu mol/(m) 2 S) the incubator temperature was controlled at 22.+ -. 1 ℃ and the concentration of algal cells in the medium was recorded daily.
As a result of this example, as shown in FIG. 1, it can be seen that the algae inoculated with the medium prepared in step 1 had no delay period observed during the activation, while the algae inoculated with the control group had been cultured for a longer period of time until day 5, and the concentration of algae in the medium prepared in step 1 was about 2 times that in the control group.
According to the experience of the past cases, the algae liquid stored in the long-term seed preservation usually needs 2-3 times of inoculation and activation to basically recover to the process of faster growth, and the algae cells can be rapidly grown after being inoculated for 1 time by adopting the culture medium prepared in the step 1 for culture. This shows that the culture medium of the application has great advantages in the activation of the seeds of the dinoflagellates 3011 such as the globes and the like, and the algae production speed is greatly improved.
Example 2
In the embodiment, culture experiments are carried out by using the seaweed seeds of the seaweed, and the specific steps are as follows:
1. according to the content of NaNO in each liter of culture medium 3 60mg，NaH 2 PO 4 8mg, ferric ammonium citrate 2.5mg, na 2 10mg of EDTA, 0.1mg of sodium 2-phosphoglycerate, 0.5mg of sodium formate, 10mg of sodium silicate, and the balance of seawater to prepare a culture medium and sterilizing the culture medium by a sterilizing pot at 115 ℃ for 15 min.
2. 50mL of Hakka Swinhonis, which was kept under low light for about 1 month, was placed in a 250L flask (150 mL of medium) in the above-described cooled medium and f/2+Si medium, and this operation was performed on an ultra clean bench.
3. Placing the inoculated algae into an illumination incubator for static culture, shaking three times a day for culture, and illuminating for 10 hours a day with illumination intensity of about 25 mu mol/(m) 2 S) the incubator temperature was controlled at 22.+ -. 1 ℃ and the concentration of algal cells in the medium was recorded daily.
The results of this example are shown in FIG. 2. As can be seen, the seaweed inoculated into the culture medium of the embodiment of the present application showed no delay period during the activation, whereas the seaweed inoculated into the control group showed a delay period of 2d, and the concentration of the seaweed of the embodiment was 3 times the concentration of the control group when cultured for 5 d. This shows that the culture medium of the application has great advantages in activating the seeds of the seaweed.
Example 3
In the embodiment, the dinoflagellate 3011 such as a ball is used for a 1-level culture test, and the specific steps are as follows:
1. according to the content of NaNO in each liter of culture medium 3 60mg，NaH 2 PO 4 8mg, ferric ammonium citrate 2.5mg, na 2 10mg of EDTA, 0.01mg of sodium 2-phosphoglycerate, 1mg of sodium formate, 5mg of sodium silicate, 0.5g of sodium diacetate, and the balance of seawater to prepare a culture medium and sterilize the culture medium by using a sterilizing pot at 115 ℃ for 15 min.
2. The operation was performed on an ultra clean bench by inoculating 300mL of the golden alga 3011 in the logarithmic growth phase to a 2L flask (1L medium) of the above-described cooled medium and f/2+Si medium, and culturing the golden alga 3011 such as a ball under the same conditions as the f/2+Si medium as a control group.
3. Placing the inoculated algae into an illumination incubator for static culture, shaking three times a day, and illuminating for 10 hours with illumination intensity of about 50 mu mol/(m) 2 S) the incubator temperature was controlled at 22.+ -. 1 ℃ and the concentration of algal cells in the medium was recorded daily.
As shown in FIG. 3, the growth rate of the algae inoculated with the medium prepared in step 1 of this example was much higher than that of the control group in the 1-stage culture, and the final concentration of 5d was about 6 times that of the control group. This shows that the culture medium of the application has great advantages in the 3011,1 grade culture of dinoflagellates such as coccidium.
Example 4
In the embodiment, the step 1 culture test is carried out by using the seaweed of the Weishihai, the method comprises the following specific steps:
1. according to the content of NaNO in each liter of culture medium 3 60mg，NaH 2 PO 4 8mg, ferric ammonium citrate 2.5mg, na 2 10mg of EDTA, 0.01mg of sodium 2-phosphoglycerate, 1mg of sodium formate, 10mg of sodium silicate, 0.5g of sodium diacetate, and the balance of seawater to prepare a culture medium and sterilize the culture medium by using a sterilizing pot at 115 ℃ for 15 min.
2. 300mL of Hachain alga of Wilkinsonii in logarithmic growth phase is put into a 2L triangular flask (1L culture medium) and the cooled culture medium and f/2+Si culture medium, and dinoflagellate 3011 such as a culture ball is used as a control group under the same condition of the f/2+Si culture medium, and the operation is carried out in an ultra-clean workbench.
3. Placing the inoculated algae into an illumination incubator for static culture, shaking three times a day, and illuminating for 10 hours with illumination intensity of about 50 mu mol/(m) 2 S) the incubator temperature was controlled at 22.+ -. 1 ℃ and the concentration of algal cells in the medium was recorded daily.
As shown in FIG. 4, the results of this example show that the growth rate of algae inoculated with the medium prepared in step 1 of this example was much higher than that of the control group in the level 1 culture, and the final concentration of 5d was about 4.5 times that of the control group. This shows that the culture medium of the application has great advantages in the class 1 culture of the seaweed.
Example 5
In the embodiment, the experiment of the seawater protein chlorella pyrenoidosa for 1-level culture is carried out by the following specific steps:
1. according to the content of NaNO in each liter of culture medium 3 60mg，NaH 2 PO 4 8mg, ferric ammonium citrate 2.5mg, na 2 10mg of EDTA, 0.01mg of sodium 2-phosphoglycerate, 1mg of sodium formate, 10mg of sodium silicate, 0.5g of sodium diacetate, and the balance of seawater to prepare a culture medium and sterilize the culture medium by using a sterilizing pot at 115 ℃ for 15 min.
2. 300mL of the marine protein nuclear chlorella in the logarithmic growth phase is inoculated into 1L of the cooled culture medium and the f/2+Si culture medium, the marine protein nuclear chlorella is cultured under the same condition of the f/2+Si culture medium as a control group, and the operation is carried out in an ultra-clean workbench.
3. Placing inoculated algaePlacing into an illumination incubator for static culture, shaking three times per day, and illuminating for 10 hours with illumination intensity of about 50 mu mol/(m) 2 S) the incubator temperature was controlled at 22.+ -. 1 ℃ and the concentration of algal cells in the medium was recorded daily.
As a result of this example, it can be seen from FIG. 5 that the algae inoculated with the medium prepared in step 1 of this example was about 10-fold higher than that of the control group in the level 1 culture. This shows that the culture medium of the application has great advantages in the 1-level culture of Chlorella pyrenoidosa.
As can be seen from the above examples-example 5, as the content of sodium 2-phosphoglycerate increases, the content of the corresponding sodium diacetate can be reduced to 0.
The same phenomenon is observed in the culture process when the rest algae, such as Phaeodactylum tricornutum, synechococcus purpureus and the like are cultured in 1-stage culture medium according to the formula shown in the table 1, the concentration of the algae is usually 3-7 times that of the culture medium of a control group (f/2+Si) in about 5 days, and sodium silicate is added in diatom and golden algae in a great amount of experimental processes to enable the growth speed to be far higher than that of the control group.
TABLE 1
2. Exploration
The present project sets up the examples to better demonstrate the amounts and effects of sodium 2-phosphoglycerate, sodium formate, sodium diacetate used in the medium, the experimental procedure is as follows.
2, 1, 2-phosphoglycerate sodium
1. According to NaNO 3 60mg，NaH 2 PO 4 8mg, ferric ammonium citrate 2.5mg, na 2 EDTA 10mg, sodium 2-phosphoglycerate concentration as shown in Table 2, sodium formate 0.5mg, sodium silicate 5mg and sea water (natural sea water, the same shall apply hereinafter) 1L were prepared as the culture medium and sterilized at 115℃for 15min in a sterilizing pot.
2. The golden alga 3011, which was kept under low light for about 2 months, was placed in a 250mL Erlenmeyer flask (150 mL of medium) and placed in the above-described cooled medium and f/2+Si, and this operation was performed on an ultra clean bench.
3. Placing the inoculated algae into an illumination incubator for static culture, shaking three times a day for culture, and illuminating for 10 hours a day with illumination intensity of about 25 mu mol/(m) 2 S) the incubator temperature was controlled at 22.+ -. 1 ℃ and the concentration of algal cells on the first day and the fifth day were compared, as shown in Table 2, it was found that when the concentration of sodium glycerophosphate was 0-0.1mg/L, the concentration of algal cells became larger as the content thereof was increased, and when the content thereof was 0.1mg/L, the concentration of algal cells 3011 had the highest concentration of cells, while the content thereof was further increased, and the concentration of algal cells did not become higher as the content thereof was increased.
TABLE 2
2.2 sodium diacetate
1. According to NaNO 3 60mg，NaH 2 PO 4 8mg, ferric ammonium citrate 2.5mg, na 2 EDTA 10mg, sodium diacetate as shown in Table 3, sodium silicate 5mg and seawater (natural seawater, the same applies hereinafter) 1L were prepared as a medium and sterilized at 115℃for 15min in a sterilizer.
2. The golden alga 3011, which was kept under low light for about 2 months, was placed in a 250mL flask (150 mL medium) and the cooled medium and f/2+Si medium, and the procedure was performed on an ultra clean bench.
3. Placing the inoculated algae into an illumination incubator for static culture, shaking three times a day for culture, and illuminating for 10 hours a day with illumination intensity of about 25 mu mol/(m) 2 S) the incubator temperature was controlled at 22.+ -. 1 ℃ and the algal cell concentrations on the first and fifth days were compared as shown in Table 3.
TABLE 3 Table 3
| Treatment group (sodium diacetate content g/L) | 1d | 5d |
| 0 | 20 | 30 |
| 0.25 | 20 | 45 |
| 0.5 | 20 | 65 |
| 0.75 | 20 | 85 |
| 1 | 20 | 105 |
| 1.25 | 20 | 55 |
| f/2+Si | 20 | 45 |
Sodium diacetate is commonly used in feed additives as a mildew inhibitor and the like. However, in the culture experiments, we have found that the concentration of algae cells is higher in the culture medium to which sodium diacetate is added than in the treatment group to which sodium diacetate is not added, and the formulation of the preferable components is designed for this purpose. It can be seen from tables 3 and 2 that the effects of sodium diacetate and sodium 2-phosphoglycerate are substantially the same, but the amounts thereof are greatly different.
2.3 sodium formate
The present inventors treated bacterial-containing algae solutions with different concentrations of sodium formate as shown in table 4. After the algae liquid is treated for 1d, the algae liquid treated by sodium formate with different concentrations is respectively taken for liquid LB culture, and the change of the bacterial concentration is detected. The algae liquid is selected from bacterial diatom algae liquid, and the results are shown in the following table:
TABLE 4 Table 4
The experimental result shows that the algae liquid can be used for inhibiting and sterilizing by adding a certain amount of sodium formate, when the concentration of the algae liquid is 0.5mg/L or more, the effect is obvious, and when the concentration is higher than 1mg/L, the effect is basically unchanged, so that the cost is increased.
The above is only a preferred embodiment of the present application, and the protection scope of the present application is not limited to the above examples, and all technical solutions belonging to the concept of the present application belong to the protection scope of the present application. It should be noted that modifications and adaptations to the present application may occur to one skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Claims (9)
1. A seawater microalgae culture medium is characterized by comprising 60-120mg of nitrogen salt, 8-12mg of phosphorus salt, 2.5-3.5mg of ferric salt and Na per liter of culture medium 2 10-12mg of EDTA, 0.01-0.1mg of sodium 2-phosphoglycerate, 0.5-1mg of sodium formate, 0-10mg of sodium silicate, 0.5-1g of sodium diacetate and the balance of seawater.
2. The seawater microalgae culture medium of claim 1, wherein: the nitrogen salt includes one or more of nitrate, ammonium salt or urea.
3. A seawater microalgae culture medium as claimed in claim 2, wherein: the nitrate comprises one or more of sodium nitrate, potassium nitrate and calcium nitrate, and the ammonium salt comprises one or more of ammonium nitrate, ammonium chloride and ammonium sulfate.
4. The seawater microalgae culture medium of claim 1, wherein: the ferric salt comprises one or more of ferric chloride, ferrous chloride, ferric sulfate, ferrous sulfate, ferric citrate and ferric ammonium citrate.
5. The seawater microalgae culture medium of claim 1, wherein: the phosphorus salt comprises one or more of sodium salt and potassium salt containing monohydrogen phosphate or dihydrogen phosphate.
6. The seawater microalgae culture medium of claim 1, wherein: the culture medium comprises potassium nitrate or NaNO 3 60-120mg，KH 2 PO 4 8-12mg, ferric ammonium citrate 2.5-3.5mg, na 2 10-12mg of EDTA, 0.01-0.1mg of sodium 2-phosphoglycerate, 0.5-1mg of sodium formate, 0-10mg of sodium silicate, 0.5-1g of sodium diacetate and the balance of seawater.
7. Use of the seawater microalgae culture medium according to any of claims 1-6 in seawater microalgae culture, characterized in that: inoculating microalgae into the seawater microalgae culture medium for culturing;
or when the primary seeds of the microalgae are activated, a small amount of preserved seeds are directly inoculated into a seawater microalgae culture medium for culture;
or when the microalgae is subjected to primary seed culture, directly inoculating the activated seeds into a seawater microalgae culture medium for culture.
8. The use according to claim 7, wherein: the seawater microalgae comprise seawater chlorella, microcystis, seawater golden algae, small diatom or rhodococcus.
9. The use according to claim 8, wherein: the small diatom comprises Chaetoceros, haematococcus, diamond-shaped algae or bone strip algae.
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