CN112553081B - Method for culturing oocyst algae by heterotrophic-autotrophic transfer two-step method - Google Patents
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Abstract
The invention relates to the technical field of microalgae cultivation, and discloses a method for cultivating oocyst algae by a heterotrophic-autotrophic transfer two-step method. The invention has the following advantages and effects: the method has the advantages that the method is characterized in that a heterotrophic-autotrophic two-step culture method is adopted originally, sodium acetate, fructose, sucrose and glucose are used as carbon sources during the heterotrophic culture, high-density culture is carried out in a fermentation tank, the culture efficiency is high, the concentration of algae liquid after fermentation culture is far higher than that of autotrophic culture, the oocyst algae obtained through the heterotrophic-autotrophic two-step culture method is high, the obtained algae liquid is high in concentration, strong in vitality, fast in self-growth speed and capable of obviously inhibiting moss, when the method is used, the canned oocyst algae are directly splashed to a culture water body, and the method has a good using effect when the method is applied to aquaculture.
Description
Technical Field
The invention relates to the technical field of microalgae cultivation, in particular to a method for cultivating oocyst algae by a heterotrophic-autotrophic two-step method.
Background
With the use of a large amount of chemical fertilizers in agricultural production and the discharge of industrial wastewater, after a large amount of nitrogen and phosphorus elements enter a water body, the environment of the aquaculture water body is gradually eutrophicated, and undesirable algae such as blue-green algae and moss in the aquaculture water body often have the capability of rapid growth, so that cyanobacterial bloom or other undesirable algae phases often appear in the aquaculture water body, which leads to the deterioration of the environment of the aquaculture water body, the poisoning of aquaculture animals and finally the failure of aquaculture.
In the prior art, beneficial algae preparations are used for intervening algae phases of aquaculture water, and the beneficial algae are directionally cultured to improve aquaculture water environment and improve aquaculture rate. In addition, the algae preparation not only has the effect of directional water fertilization, but also has the functions of increasing dissolved oxygen in water and absorbing ammonia nitrogen and nitrite, so that the algae preparation is widely applied to aquaculture. The oocysts are relatively stable green algae, can keep the phenomenon of algae falling for 40-50 days, and have relatively strong capacity of absorbing ammonia nitrogen and nitrite, so the oocysts are the microalgae for well improving water quality strips and cultivating water algae phases.
In the prior art, oocysts are used as beneficial algae to improve the water environment, for example, chinese patent application with application publication No. CN106719289A discloses a method for regulating and controlling water quality in aquaculture by using the oocysts, which has exclusivity on vibrio and blue algae. The application publication number of CN110547237A is Chinese patent application, which discloses a method for preventing and controlling cyanobacterial bloom in a shrimp pond by using oocyst algae.
However, in the practical application process of the oocysts, the following problems exist, namely the culture mode of the oocysts is mostly autotrophic illumination culture, the culture efficiency is low, and the concentration of the cultured algae solution is low, so that the popularization and the use of the oocysts are influenced. Secondly, the harmful algae such as vibrio, blue algae and the like can be inhibited by using the oocysts at present, but no oocysts aiming at inhibiting the growth of moss are domesticated.
Disclosure of Invention
The invention aims to provide a method for culturing oocyst algae by a heterotrophic-autotrophic two-step method.
The technical purpose of the invention is realized by the following technical scheme: a method for culturing oocyst algae by a heterotrophic-autotrophic two-step method comprises the following steps of S1, performing heterotrophic culture, inoculating a seed solution of the oocyst algae into a fermentation tank, taking ammonium salt as a nitrogen source, taking sodium acetate, fructose, sucrose and glucose as carbon sources, and controlling the carbon-nitrogen ratio to be 10:1, monitoring the pH value of the fermentation tank in the culture process, and adjusting by using ammonia water to maintain the pH value in the fermentation tank within the range of 7.5 +/-0.2, and finishing fermentation when the fermentation biomass reaches 35000-50000 mg/L; s2, autotrophic culture, namely inoculating the oocyst algae solution obtained in the S1 into a photoreactor as seed solution, selecting a special culture medium for the oocyst algae, inoculating the special culture medium according to the inoculation amount of 1-3 per mill, sterilizing culture water by using sodium hypochlorite, performing light culture, using the light to be 3000-6000 lux, and canning the cultured biomass until the biomass reaches 400-500 mg/L.
By adopting the technical scheme, the high-concentration oocyst algae is obtained in the step S1, and the special culture medium for the oocyst algae is a special culture medium for culturing the high-concentration oocyst algae.
The invention is further configured as follows: the special culture medium for the oocyst algae comprises a solution A and a solution B, wherein the formula of the solution A comprises NaNO 3 50-180g/L，CH 4 N 2 O 20-50g/L，NaH 2 PO 4 ·2H 2 O2-6 g/L and EDTA disodium 3-5g/L; feCl 3 ·6H 2 O 2-3g/L，C 6 H 8 O 7 5-40g/L; the formula of the solution B comprises a solution C 6 H 8 FeNO 7 3-8g/L，CaCl 2 ·2H 2 O20-50g/L，H 3 BO 3 2-3g/L，MnCl 2 ·4H 2 O 0.5-3g/L，ZnSO 4 ·7H 2 O 0.2-0.6g/L，Na 2 MoO 4 ·2H 2 O 0.3-0.8g/L，CuSO 4 ·5H 2 O 0.5-0.8g/L，Co(NO 3 ) 2 ·6H 2 0.3-0.6g/L of O, 0.1-0.3g/L of vitamin B, 0.005-0.001g/L of vitamin B and 0.003-0.005g/L of biotin.
By adopting the technical scheme, CH 4 N 2 O is urea, C 6 H 8 O 7 Is citric acid, C 6 H 8 FeNO 7 Is ferric ammonium citrate, H 3 BO 3 Is boric acid.
The invention is further provided with: in S1, the fermentation is ended when the biomass of fermentation reaches 40000 mg/L.
The invention is further provided with: and S2, culturing until the biomass reaches 400mg/L, and canning.
The invention is further provided with: s1, the preparation method of the oocyst algae seed solution comprises the following steps of (1) inoculating oocyst algae seeds on a flat plate, and culturing at the temperature of 25-35 ℃ for 10 days to form algae colonies; step (2), selecting algae to fall into 20-50mL of culture medium for shake culture at the temperature of 25-35 ℃ and the rotating speed of 50-150rpm/min, and culturing for 4-6 days to obtain first-stage seed liquid; transferring the primary seed solution into 300mL of culture medium, culturing at 25-35 ℃ and at the rotation speed of 50-150rpm/min, performing shake culture, and culturing for 4-6 days to obtain a secondary seed solution; and (4) transferring the secondary seed solution into a 3L culture medium, wherein the culture temperature is 25 ℃, the rotation speed is 50-150rpm/min, performing shake culture, and taking the seed solution cultured for 4-6 days as a fermentation tank to culture the algae.
The invention is further provided with: in S1, the inoculation amount of the oocyst seed solution is 2.5-20%.
The invention is further provided with: in S1, the inoculation amount of the oocyst algae seed liquid is 10 percent
The beneficial effects of the invention are:
1. in the prior art, the oocyst algae is cultured by adopting light autotrophic low-density culture, the culture concentration is low, and the efficiency is low. The method has the advantages that the culture is creatively carried out by adopting a heterotrophic-autotrophic two-step method, sodium acetate, fructose, sucrose and glucose are used as carbon sources during the heterotrophic culture, the high-density culture is carried out in the fermentation tank, the culture efficiency is high, and the concentration of the algae liquid after the fermentation culture is far higher than that of the autotrophic culture.
2. When heterotrophic transformation autotrophic transformation is carried out, a culture medium special for oocyst algae is designed in a targeted manner, through setting of all components and concentration and assisted with induction of 3000lux-6000lux with high illumination, chlorophyll in the oocyst algae is rapidly synthesized, the cell morphology is rapidly recovered to the autotrophic morphology, the obtained oocyst algae is high in activity and high in autotrophic growth speed, and the culture medium has a better using effect when being applied to aquaculture.
3. In the process of co-culture experiment of the oocyst algae and the moss, the phenomenon that the structure of the moss cell is changed and part of the moss cells are dissolved is observed, and the oocyst algae cell is analyzed to generate secretion for inhibiting the growth of the moss through induction.
4. The oocyst algae cultured by the heterotrophic-autotrophic two-step method has high culture efficiency, the obtained algae liquid has high concentration, strong activity and fast self-growth speed, and has obvious inhibition effect on moss.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph showing the growth of each example and comparative example in the experiment.
FIG. 2 shows the moss morphology of the experiment showing inhibition of cocultivation of oocysts with moss.
FIG. 3 shows microscopic morphology of moss from the experiment of inhibition of co-culture of oocysts with moss.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without inventive step, shall fall within the scope of protection of the present invention.
Example 1: the method for culturing the oocyst algae by the heterotrophic-autotrophic two-step method comprises the following steps of S1, heterotrophic culture and preparation of oocyst algae seed solution, wherein the preparation method comprises the following steps: inoculating oocyst algae seeds on a flat plate, culturing at the temperature of 25-35 ℃ for 10 days to form algae colonies; step (2), selecting algae to fall into 40mL of culture medium for shake culture at the temperature of 25-35 ℃ and the rotating speed of 100rpm/min, and culturing for 4-6 days to obtain first-stage seed liquid; transferring the primary seed solution into 300mL of culture medium, culturing at 25-35 ℃ at a rotation speed of 100rpm/min, performing shake culture, and culturing for 5 days to obtain a secondary seed solution; and (4) transferring the secondary seed solution into a 3L culture medium, wherein the culture temperature is 25 ℃, the rotation speed is 100rpm/min, performing shake culture, and taking the seed solution cultured for 5 days as a fermentation tank to culture the algae.
Inoculating the oocyst algae seed solution into a fermentation tank, wherein the inoculation amount of the oocyst algae seed solution is 10%. Taking ammonium salt as a nitrogen source, taking sodium acetate, fructose, sucrose and glucose as carbon sources, wherein the carbon-nitrogen ratio is 40:1, monitoring the pH value of the fermentation tank in the culture process, and adjusting by using ammonia water to maintain the pH value in the fermentation tank within the range of 7.5 +/-0.2, and finishing fermentation when the fermentation biomass reaches 40000 mg/L;
s2, autotrophic culture, namely inoculating the oocyst algae solution obtained in the S1 into a photoreactor as seed solution, selecting a special culture medium for the oocyst algae, inoculating the seed solution according to the inoculation amount of 2 per mill, sterilizing the culture medium with water through sodium hypochlorite, performing illumination culture, and canning when the biomass reaches 400mg/L and the illumination is 4000 lux.
The special culture medium for the oocyst algae comprises a solution A and a solution B, wherein solutes and concentrations of the solution A are as follows: naNO 3 160g/L，CH 4 N 2 O 40g/L，NaH 2 PO 4 2H2O 5g/L, EDTA disodium 4g/L; feCl 3 ·6H 2 O 3g/L，C 6 H 8 O 7 6g/L。
The solute and concentration of solution B are as follows: c 6 H 8 FeNO 7 6g/L，CaCl 2 ·2H 2 O 40g/L，H 3 BO 3 3g/L，MnCl 2 ·4H 2 O 2g/L，ZnSO 4 ·7H 2 O 0.5g/L，Na 2 MoO 4 ·2H 2 O 0.6g/L，CuSO 4 ·5H 2 O 0.6g/L，Co(NO 3 ) 2 ·6H 2 0.5g/L of O, 0.2g/L of vitamin B, 0.001g/L of vitamin B and 0.005g/L of biotin.
CH 4 N 2 O is urea, C 6 H 8 O 7 Is citric acid, C 6 H 8 FeNO 7 Is ferric ammonium citrate, H 3 BO 3 Is boric acid.
The volume ratio of the solution A to the solution B is 3.
Example 2: the method for culturing the oocyst algae by the heterotrophic-autotrophic two-step method comprises the following steps of S1, heterotrophic culture and preparation of oocyst algae seed solution, wherein the preparation method comprises the following steps: inoculating oocyst algae seeds on a flat plate, and culturing at the temperature of 25-35 ℃ for 10 days to form algae colonies; picking algae to fall into a 20mL culture medium for shake culture at the culture temperature of 25-35 ℃ and the rotation speed of 50rpm/min, and culturing for 4 days to obtain a primary seed solution; transferring the primary seed solution into 300mL of culture medium at the temperature of 25-35 ℃ at the rotating speed of 50rpm/min, performing shake culture, and culturing for 4 days to obtain a secondary seed solution; and (4) transferring the secondary seed solution into a 3L culture medium, carrying out shake culture at the culture temperature of 25 ℃ and the rotation speed of 50rpm/min, and using the seed solution cultured for 4 days as a fermentation tank to culture algae.
Inoculating the oocyst algae seed solution into a fermentation tank, wherein the inoculation amount of the oocyst algae seed solution is 2.5%. Taking ammonium salt as a nitrogen source, taking sodium acetate, fructose, sucrose and glucose as carbon sources, wherein the carbon-nitrogen ratio is 10;
s2, autotrophic culture, namely inoculating the oocyst algae solution obtained in the S1 into a photoreactor as seed solution, selecting a special culture medium for the oocyst algae, inoculating the culture medium according to 1 per thousand inoculation amount, sterilizing the culture medium with water through sodium hypochlorite, culturing the culture medium by illumination of 3000lux, and canning the culture medium until the biomass reaches 400 mg/L.
The special culture medium for the oocyst algae comprises a solution A and a solution B, wherein the formula of the solution A is as follows: naNO 3 50g/L，CH 4 N 2 O 20g/L，NaH 2 PO 4 ·2H 2 O2 g/L, EDTA disodium 3g/L; feCl3.6H 2 O 2g/L,C 6 H 8 O 7 40g/L。
The solute and concentration of the solution B are as follows: c 6 H 8 FeNO 7 3g/L，CaCl 2 ·2H 2 O 20g/L，H 3 BO 3 2g/L，MnCl 2 ·4H 2 O 0.5g/L，ZnSO 4 ·7H 2 O 0.2g/L，Na 2 MoO 4 ·2H 2 O 0.3g/L，CuSO 4 ·5H 2 O 0.5g/L，Co(NO 3 ) 2 ·6H 2 0.3g/L of O, 0.1g/L of vitamin B, 0.005g/L of vitamin B and 0.003g/L of biotin.
CH 4 N 2 O is urea, C 6 H 8 O 7 Is citric acid, C 6 H 8 FeNO 7 Is ferric ammonium citrate, H 3 BO 3 Is boric acid.
The volume ratio of the solution A to the solution B is 3.
Example 3: the method for culturing the oocyst algae by the heterotrophic-autotrophic two-step method comprises the following steps of S1, heterotrophic culture and preparation of oocyst algae seed solution, wherein the preparation method comprises the following steps: inoculating oocyst algae seeds on a flat plate, culturing at the temperature of 25-35 ℃ for 10 days to form algae colonies; picking algae to fall into a 50mL culture medium for shake culture at the temperature of 25-35 ℃ at the rotating speed of 150rpm/min, and culturing for 6 days to obtain a primary seed solution; transferring the primary seed solution into 300mL of culture medium, culturing at 25-35 ℃ at a rotation speed of 150rpm/min by shaking, and culturing for 6 days to obtain a secondary seed solution; and (4) transferring the secondary seed solution into a 3L culture medium, wherein the culture temperature is 25 ℃, the rotation speed is 50-150rpm/min, performing shake culture, and taking the seed solution cultured for 6 days as a fermentation tank to culture the algae.
Inoculating the oocyst algae seed solution into a fermentation tank, wherein the inoculation amount of the oocyst algae seed solution is 20%. Taking ammonium salt as a nitrogen source, taking sodium acetate, fructose, sucrose and glucose as carbon sources, wherein the carbon-nitrogen ratio is 50:1, monitoring the pH value of the fermentation tank in the culture process, and adjusting by using ammonia water to maintain the pH value in the fermentation tank within the range of 7.5 +/-0.2, and finishing fermentation when the fermentation biomass reaches 40000 mg/L;
s2, autotrophic culture, namely inoculating the oocyst algae solution obtained in the S1 into a photoreactor as seed solution, selecting a special culture medium for the oocyst algae, inoculating the culture medium according to the inoculation amount of 3 per mill, sterilizing the culture medium with water through sodium hypochlorite, performing light culture, using the light to be 6000lux, and canning the culture medium until the biomass reaches 400 mg/L.
The special culture medium for the oocyst algae comprises a solution A and a solution B, wherein solutes and concentrations of the solution A are as follows: naNO 3 180g/L，CH 4 N 2 O 50g/L，NaH 2 PO 4 ·2H 2 O6 g/L and EDTA disodium 5g/L; feCl3.6H 2 O 3g/L，C 6 H 8 O 7 30g/L。
The solute and concentration of solution B are as follows: c 6 H 8 FeNO 7 8g/L，CaCl 2 ·2H 2 O 50g/L，H 3 BO 3 3g/L，MnCl 2 ·4H 2 O 3g/L，ZnSO 4 ·7H 2 O 0.6g/L，Na 2 MoO 4 ·2H 2 O 0.8g/L，CuSO 4 ·5H 2 O 0.8g/L，Co(NO 3 ) 2 ·6H 2 0.6g/L of O, 0.3g/L of vitamin B, 0.001g/L of vitamin B and 0.005g/L of biotin.
CH 4 N 2 O is urea, C 6 H 8 O 7 Is citric acid, C 6 H 8 FeNO 7 Is ferric ammonium citrate, H 3 BO 3 Is boric acid.
Example 4: the difference between the method for culturing the oocyst algae by the heterotrophic-autotrophic two-step method and the embodiment 2 is that in S1, the fermentation is finished when the fermentation biomass reaches 35000 mg/L. In S2, the biomass is cultured until the biomass reaches 500mg/L by using the illumination of 5000 and then is canned.
Example 5: the difference between the heterotrophic autotrophic two-step culture method of oocyst algae and the example 3 is that the fermentation is finished when the fermentation biomass reaches 50000mg/L in S1. In S2, the illumination was 5000.
Experiment one was conducted to determine the growth curve during autotrophic growth in each example, and the control group was inoculated with conventional oocysts to determine the growth curve.
As shown in FIG. 1, the growth rate of the experimental group is significantly better than that of the control group, indicating strong activity and fast growth speed.
Experiment of Co-culture inhibition of oocysts and lichen
Cleaning and draining moss with clear water, spin-drying water by using a centrifuge at 4000rpm, cleaning the moss with the clear water for 2 times, accurately weighing 0.2g of moss in each 1L triangular flask, and adding 500mL of water. In the experimental group, 0.1g of each of the oocysts obtained in example 1, example 2 and example 3 was weighed and put into three triangular flasks. The blank group was prepared by adding 5g of oocyst algae cultured in a conventional medium (BG 11) to a flask as a control. And placing illumination culture on the illumination culture shelf. After 7 days, the moss is filtered out, the moss is dried by a centrifuge at 4000rpm, and is washed by clean water for 2 times, and then the moss is weighed, observed in shape and observed under a microscope.
Test results and analysis: as shown in Table 1, the experimental group has a relatively obvious inhibition effect on moss, and the weight of the moss is obviously smaller than that of the moss in the blank group. As shown in FIG. 2, the form of the moss in the experimental group was significantly changed, and the moss turned yellow and began to decay. As shown in fig. 3, the moss cells in the blank group had a robust structure, deep chlorophyll color, and uniform and full distribution, and after high-density culture of algae in the experimental group, the moss cells all had a changed structure, reduced chlorophyll, and non-uniform distribution, some cells became shriveled and easily broken, the cell edges were unclear, and some cells even appeared dissolved. The invention shows that the oocyst algae prepared by the invention can secrete substances to inhibit the growth of the moss and has obvious inhibition effect on the moss through high illuminance and induction and domestication of the culture medium special for the oocyst algae.
TABLE 1 weighing results after coculture of oocysts and lichens
Claims (7)
1. The method for culturing the oocyst algae by the heterotrophic-autotrophic two-step method is characterized by comprising the following steps: comprises the following steps
S1, heterotrophic culture, namely inoculating the oocyst algae seed solution into a fermentation tank, taking ammonium salt as a nitrogen source, taking sodium acetate, fructose, sucrose and glucose as carbon sources, and adjusting the carbon-nitrogen ratio to be 10-50: 1, monitoring the pH value of the fermentation tank in the culture process, and adjusting by using ammonia water to maintain the pH value in the fermentation tank within the range of 7.5 +/-0.2, and finishing fermentation when the fermentation biomass reaches 35000-50000 mg/L;
s2, autotrophic culture, namely inoculating the oocyst algae solution obtained in the S1 serving as seed solution into a photoreactor, selecting a special culture medium for the oocyst algae, inoculating the culture medium according to the inoculation amount of 1-3 per thousand, sterilizing culture water by using sodium hypochlorite, performing illumination culture, using the illumination of 3000-6000 lux, and canning when the biomass reaches 400-500 mg/L.
2. The method of claim 1, wherein the method comprises: the special culture medium for the oocyst algae comprises a solution A and a solution B, wherein the formula of the solution A comprises NaNO 3 50-180g/L，CH 4 N 2 O 20-50g/L，NaH 2 PO 4 ·2H 2 O2-6 g/L3-5g/L of EDTA disodium; feCl 3 ·6H 2 O 2-3g/L；C 6 H 8 O 7 5-40g/L; the formula of the solution B comprises 6 H 8 FeNO 7 3-8g/L，CaCl 2 .2H 2 O20-50g/L，H 3 BO 3 2-3g/L，MnCl 2 ·4H 2 O 0.5-3g/L，ZnSO 4 ·7H 2 O 0.2-0.6g/L，Na 2 MoO 4 .2H 2 O 0.3-0.8g/L，CuSO 4 .5H 2 O 0.5-0.8g/L，Co(NO 3 ) 2 .6H 2 0.3-0.6g/L of O, 0.1-0.3g/L of vitamin B, 0.005-0.001g/L of vitamin B and 0.003-0.005g/L of biotin.
3. The method of claim 1, wherein the method comprises: in S1, the fermentation is finished when the fermentation biomass reaches 40000 mg/L.
4. The method of claim 1, wherein the step of culturing oocysts is performed in S2 until the biomass reaches 400 mg/L.
5. The method of claim 1, wherein the method comprises: s1, the preparation method of the oocyst algae seed solution comprises the following steps of (1) inoculating oocyst algae seeds on a flat plate, and culturing at the temperature of 25-35 ℃ for 10 days to form algae colonies; picking algae to fall into a culture medium of 20-50mL for shake culture at the culture temperature of 25-35 ℃ and the rotation speed of 50-150rpm/min, and culturing for 4-6 days to obtain a first-stage seed solution; transferring the primary seed solution into 300mL of culture medium at the temperature of 25-35 ℃ and at the rotating speed of 50-150rpm/min, performing shake culture, and culturing for 4-6 days to obtain a secondary seed solution; and (4) transferring the secondary seed solution into a 3L culture medium, performing shake culture at the culture temperature of 25 ℃ and the rotation speed of 50-150rpm/min, and culturing the algae seeds by using the seed solution cultured for 4-6 days as a fermentation tank.
6. The method of claim 5, wherein the method comprises: in S1, the inoculation amount of the oocyst seed solution is 2.5-20%.
7. The method of claim 6, wherein the method comprises: in S1, the inoculation amount of the oocyst algae seed liquid is 10%.
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