A method for inhibiting chlorella protothecoides infection in cultivation of haematococcus pluvialis

Abstract

The present application relates to a method for inhibiting chitin infection in Haematococcus cultivation, and belongs to the technical field of Haematococcus cultivation. The present application adds caspofungin and beta-mannanase to Haematococcus infected with chitin, and controls the pH value of the algal liquid to be between 8.0-9.5. Whether in the green alga cultivation stage or in the red alga cultivation stage, the present application can effectively inhibit chitin, inhibit the synthesis of the chitin cell wall, control the proliferation and parasitism of chitin from the source, reduce the low astaxanthin content and cell death of Haematococcus caused by chitin infection, reduce the cultivation risk, and improve the astaxanthin yield after chitin infection.

Description

Technical Field

[0001] This invention belongs to the field of Haematococcus pluvialis cultivation technology, specifically, it relates to a method for inhibiting chytrid fungal infection in Haematococcus pluvialis cultivation. Background Technology

[0002] Haematococcus pluvialis contains 10-40 mg of astaxanthin, making it the organism with the highest natural astaxanthin content in nature. Furthermore, the astaxanthin in Haematococcus pluvialis has a 100% levorotatory structure, which is stable and consistent with the structure of astaxanthin required by humans and animals, facilitating absorption and maximizing its various biological benefits. The cultivation of Haematococcus pluvialis involves first cultivating algal strains indoors, then expanding the green algae culture in an outdoor airlift photobioreactor (PB bag) (green algae cultivation stage), and finally introducing red algae culture medium into a tubular photobioreactor (GP pipeline) for large-scale stress cultivation (red algae cultivation stage). During the large-scale outdoor cultivation of Haematococcus pluvialis, chytrid fungus infection is common. Chytrid fungus is an obligate parasitic pathogen of Haematococcus pluvialis. When the green algae spores of Haematococcus pluvialis are infected by chytrid fungus during large-scale cultivation, it will lead to a large-scale outbreak of chytrid fungus infection when the algae are cultured in GP pipelines under stress. Once the infection rate of chytrid fungus in the red algae cultured in GP pipelines exceeds 50%, all algal cells will die within 3-5 days, resulting in no harvest from the large amount of manpower, material resources and algal strains invested in the early stage of cultivation, which is a significant loss for the enterprise.

[0003] Although chytridactylus infection has a significant impact on the large-scale cultivation of Haematococcus pluvialis, research on chytridactylus is currently extremely limited. Studies have shown that chytridactylus can attach to the surface of Haematococcus pluvialis cells, using its hyphae to penetrate the cell wall, absorb nutrients from the host cell for growth, and ultimately lead to the death of the host cell. However, research on inhibiting chytridactylus infection is scarce. Some studies have shown that surfactants such as sodium dodecylbenzenesulfonate, sodium dodecyl sulfate, and primary alcohol ethoxylates can inhibit chytridactylus infection in Haematococcus pluvialis. However, a problem in practical application is that Haematococcus pluvialis, as an algae, needs to be cultivated in an aquatic environment. The dissolution of surfactants in the water used for cultivation leads to the generation of large amounts of foam. Because algal cells are extremely small, the large amount of foam generated after adding surfactants causes algal cells to adhere and become encapsulated, overflowing from the culture container and leaking out, resulting in a significant loss of algal cells and ultimately a very low harvest. Studies have also shown that using acidic conditions (pH 4) instead of the neutral pH culture medium used in normal cultivation can significantly inhibit Chytridacella infection. However, in practical applications, since Haematococcus pluvialis is a freshwater algae colony, its green algae stage is adapted to a neutral to slightly alkaline environment (7.5–8.0). After using acidic conditions (pH 4) to cultivate Haematococcus pluvialis, the algal cells all lyseed and died, making effective cultivation impossible.

[0004] Currently, there is no effective and practical method for treating chytrid fungal infection during Haematococcus pluvialis cultivation. Summary of the Invention

[0005] To address the problems existing in the background technology, this invention provides a method for inhibiting chytrid fungus infection in Haematococcus pluvialis cultivation. By adding caspofungin and β-mannanase to the Haematococcus pluvialis algal solution, chytrid fungus infection can be effectively inhibited, reducing the low astaxanthin content and cell death in Haematococcus pluvialis caused by chytrid fungus infection, reducing cultivation risks, and increasing astaxanthin production after chytrid fungus infection. This method can have a significant inhibitory effect on chytrid fungus in both green algae cultivation and red algae cultivation stages.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] Adding caspofungin and β-mannanase to the algal solution of Haematococcus pluvialis infected with Chytridium can inhibit Chytridium and reduce its impact on Haematococcus pluvialis.

[0008] Furthermore, the concentration of caspofungin in the algal solution was greater than 1 ppm, and the concentration of β-mannanase in the algal solution was greater than 1 ppm.

[0009] Furthermore, the concentration of caspofungin in the algal solution is 1-5 ppm, and the concentration of β-mannanase in the algal solution is 1-6 ppm.

[0010] Furthermore, the concentration ratio of caspofungin to β-mannanase is 1:1-2.

[0011] Furthermore, the pH of the algal solution should be controlled between 8.0 and 9.5.

[0012] Furthermore, the pH value of the algal solution is controlled by introducing carbon dioxide into the solution.

[0013] Furthermore, the Haematococcus pluvialis mentioned are Haematococcus pluvialis green algae cultured in outdoor PB bags and Haematococcus pluvialis red algae cultured in GP pipes.

[0014] Application of caspofungin and β-mannanase in the inhibition of Chytridacobacterium.

[0015] Application of caspofungin and β-mannanase in inhibiting chytrid fungi during Haematococcus pluvialis cultivation.

[0016] The beneficial effects of this invention are:

[0017] This invention can effectively inhibit the impact of chytrid fungi on Haematococcus pluvialis cultivation, reduce the low astaxanthin content and cell death in Haematococcus pluvialis caused by chytrid fungi infection. Validated through large-scale Haematococcus pluvialis cultivation practice, in the green algae stage, the chytrid fungi infection rate of green spore cells can be suppressed to below 2% in PB bags without affecting the division and proliferation of green mobile cells. In the red algae stage, the chytrid fungi infection rate can be suppressed to below 1% within 12 days after Haematococcus pluvialis transforms into red spores in GP pipes, effectively reducing the cultivation risk of Haematococcus pluvialis and increasing astaxanthin production after chytrid fungi infection.

[0018] This invention is not only simple and easy to operate with low cost, but also effectively inhibits chytrids and reduces their impact on Haematococcus pluvialis. Furthermore, the pH value controlled by the method used in this invention will not adversely affect the cultivation of Haematococcus pluvialis, and can greatly reduce the chytrid infection rate of Haematococcus pluvialis. The final dry weight of algal powder and its astaxanthin content are not significantly different from those of the uninfected group, which greatly reduces the cost of pollution control for Haematococcus pluvialis cultivation enterprises.

[0019] The caspofungin and β-mannanase used in this invention are both readily available chemical reagents. In addition, they are used in small quantities, are low in cost, are easy to use, and have significant effects, which is of great significance for the cultivation of Haematococcus pluvialis. Attached Figure Description

[0020] Figure 1 This is a microscopic image (×100) showing the inhibitory effect of caspofungin and β-mannanase on Chytrids in PB bags cultured during the green algae stage.

[0021] Figure 2 This is a microscopic image (×40) showing the inhibitory effect of caspofungin and β-mannanase on Chytrids in GP pipes during the red algae cultivation stage of the present invention.

[0022] Remark:

[0023] Figure 1 In the study, the normal control group consisted of Haematococcus pluvialis cultured normally in the green algae stage in PB bags without the addition of Chytridium-infected algae solution. The blank control group consisted of Haematococcus pluvialis cultured in the green algae stage in PB bags with the addition of Chytridium-infected algae solution but without other treatment reagents. The drug-treated group consisted of Haematococcus pluvialis cultured in the green algae stage in PB bags with the addition of Chytridium-infected algae solution, along with 2 ppm caspofungin and 3 ppm β-mannanase. The arrows indicate Chytridium-infected green spore cells.

[0024] Figure 2In the study, the normal control group consisted of Haematococcus pluvialis cultured normally in the red algae stage in GP pipelines without the addition of Chytridium-infected algae solution. The blank control group consisted of Haematococcus pluvialis cultured in the red algae stage in GP pipelines with the addition of Chytridium-infected algae solution but without the addition of other treatment reagents. The drug treatment group consisted of Haematococcus pluvialis cultured in the red algae stage in GP pipelines with the addition of Chytridium-infected algae solution, along with the addition of 2 ppm caspofungin and 3 ppm β-mannanase. Detailed Implementation

[0025] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are all within the scope of protection of this invention.

[0026] The method for inhibiting chytrid fungal infection in Haematococcus pluvialis cultivation according to the present invention includes the following steps (taking the green algae cultivation stage as an example):

[0027] (1) Haematococcus pluvialis was cultured in outdoor PB bags and its chytrid contamination was monitored daily.

[0028] (2) After chytrid contamination occurs, add caspofungin and β-mannanase to the PB bag, and adjust the carbon dioxide ventilation daily to keep the pH of the algal solution in the PB bag constant between 8.0 and 9.5.

[0029] In step (2), the concentrations of caspofungin and β-mannanase in the algal solution were both greater than 1 ppm. After the chytrids were detected, they were added to the algal solution at concentrations of 1-5 ppm and 1-6 ppm, respectively.

[0030] The method for inhibiting chytrid fungal infection in the cultivation of Haematococcus pluvialis according to the present invention includes the following steps (taking the red algae cultivation stage as an example):

[0031] (1) Haematococcus pluvialis was cultured in outdoor GP pipes and its chytrid contamination was monitored daily.

[0032] (2) After confirming the presence of Chytridacobacter contamination, add caspofungin and β-mannanase to the pipeline, and adjust the carbon dioxide ventilation daily to keep the pH of the algal solution in the GP pipeline constant between 8.0 and 9.5.

[0033] In step (2), the concentrations of caspofungin and β-mannanase in the algal solution were both greater than 1 ppm. After the chytrids were detected, they were added to the algal solution at concentrations of 1-5 ppm and 1-6 ppm, respectively.

[0034] The effect of *Chytridactylum* on *Haematococcus pluvialis* is mainly through attachment to the cell surface of *Haematococcus pluvialis*, using its hyphae to penetrate the cell wall, absorbing nutrients from the *Haematococcus pluvialis* cells for growth, ultimately leading to the death of *Haematococcus pluvialis*. During the parasitic process of *Chytridactylum*, it needs to synthesize large amounts of glucan and mannan required for fungal cell wall formation. Adding caspofungin can inhibit 1,3-β-D-glucan synthase. Since β-mannanase's substrate is mannan, the combined use of these two can effectively inhibit fungal cell wall synthesis. By inhibiting *Chytridactylum* cell wall synthesis, the proliferation and parasitism of *Chytridactylum* can be controlled at its source.

[0035] To illustrate the present invention more clearly, the following embodiments will be described in detail.

[0036] Example 1 (Green Algae Cultivation Stage)

[0037] The method for preventing contamination of Haematococcus pluvialis in the green algae stage and Chytrid fungi in PB bags includes the following steps:

[0038] (1) Add Haematococcus pluvialis green algae solution and BG11 culture medium to an outdoor PB bag, then add filtered water to the calibration volume. The Haematococcus pluvialis green motile cells are introduced at a rate of 2×10⁻⁶. 4 The cells were cultured at a density of approximately 3-5 × 10⁶ cells / ml the next day, and the number of green motile cells was approximately 3-5 × 10⁶. 4 PB bags containing 10 cells / ml were used as experimental subjects, with a density of 10 × 10⁻⁶ red immobile cells. 4Haematococcus pluvialis infection solution with a concentration of 1 / ml and an infection rate of 50% was added to the PB bags of each experimental group. At this time, the total infection rate of Haematococcus pluvialis chytrid fungus in the PB bags of the experimental groups was about 0.6%-1.0%. The algal solution without chytrid fungus infection solution was used as the normal control group. (2) Add different concentrations of caspofungin and β-mannanase to the PB bags of each experimental group, and use the control group without adding any reagents to control chytrids as a blank control group; (3) Monitor the chytrid contamination, number of motile cells and number of dividing cells daily; (4) Detect the pH value of the algal solution in the PB bags daily, and adjust the carbon dioxide and air ventilation to keep the pH value of the algal solution constant between 8.0 and 9.5; (5) Take the algal solution infected with chytrids as day 0, and then culture for 6 days. The entire process is 8 days in PB bags. Detect the chytrid infection rate, number of motile cells and number of dividing cells of green spore cells in the PB bags on the last day; (6) Then, the algal solution in the 6 PB bags of the same experimental group is connected to a GP pipe with a volume of 3000L for culture. There are three GP pipes in each experimental group. After 20 days of culture, the chytrid infection rate of red spore cells in the GP pipes is detected before harvest. Harvest the red spores of Haematococcus pluvialis and dry them into algal powder. Detect the dry weight of algal powder and the content of astaxanthin. The detection results are shown in Table 1. Table 1 shows that the infection rates of green spore cells in PB bags and red spore cells in GP pipes in the experimental groups with added caspofungin and β-mannanase were significantly lower than those in the blank control group. Furthermore, the number of motile and dividing cells in the experimental groups with added caspofungin and β-mannanase in PB bags were not significantly different from those in the normal control group. In addition, the final harvested algal powder dry weight and astaxanthin content were significantly higher than those in the blank control group. Among them, the infection rate, algal powder dry weight and astaxanthin content of the group with added 2 ppm caspofungin + 3 ppm β-mannanase were significantly different from those of the blank control group.

[0039] Table 1 Comparison of the effects of caspofungin and β-mannanase concentrations on green algae cultivation

[0040]

[0041]

[0042] Compared with the blank control group, **P<0.01, ***P<0.001; compared with the normal control group, ##P<0.01, ###P<0.001

[0043] Microscopic images showing the inhibition of Chytridium in PB bags are as follows. Figure 1 ,from Figure 1It can be seen that: there was no significant difference in the infection rate of Chrystridium perfringens between the blank control group and the drug-treated group on day 0, while the infection rate of Chrystridium perfringens in the blank control group was significantly higher than that in the drug-treated group on day 6; there was no significant difference in the number of motile cells between the normal control group and the drug-treated group on day 0, and there was still no significant difference in the number of motile cells between the normal control group and the drug-treated group on day 6, indicating that the drug treatment effectively inhibited Chrystridium perfringens infection.

[0044] Example 2 (Green Algae Cultivation Stage)

[0045] The method for preventing contamination of Haematococcus pluvialis in the green algae stage and Chytrid fungi in PB bags includes the following steps:

[0046] (1) Add Haematococcus pluvialis green algae solution and BG11 culture medium to an outdoor PB bag, then add filtered water to the calibration volume, so that the Haematococcus pluvialis green motile cells grow at a rate of 2×10⁻⁶. 4 The cells were cultured at a density of approximately 3-5 × 10⁶ cells / ml the next day, and the number of green motile cells was approximately 3-5 × 10⁶. 4 Using PB bags containing 100 cells / ml as the experimental subject, and 100 ml of bags containing only red, immobile cells at a density of 100 × 10⁻⁶ cells / ml, the experiment was conducted. 4 Haematococcus pluvialis infection solution with a concentration of 1 / ml and an infection rate of 50% was added to the PB bags of each experimental group. At this time, the total infection rate of Haematococcus pluvialis chytrid fungus in the PB bags of the experimental groups was about 0.6%-1.0%. The algal solution without chytrid fungus infection was used as the normal control group. (2) Caspofungin and β-mannanase were used alone and in combination in the PB bags of each experimental group as different experimental groups. At the same time, the control group without adding any reagents to control chytrid fungus was used as the blank control group. (3) The chytrid fungus contamination, number of motile cells and number of dividing cells were monitored daily. (4) The pH value of the algal solution in the PB bags was detected daily, and the carbon dioxide and air ventilation were adjusted to ensure that the pH value was constant between 8.0 and 9.5. (5) The chytrid fungus-infected algal solution was taken as day 0, followed by 6 days of culture, and a total of 8 days of culture in PB bags. The chytrid fungus infection rate, number of motile cells and number of dividing cells in the green spore cells of the PB bags were detected on the last day. (6) Subsequently, the algal solutions from 6 PB bags of the same experimental group were inoculated into a set of 3000L GP pipes for culture. Each experimental group had three sets of GP pipes. After 20 days of culture, the chytrid fungus infection rate of red spore cells in the GP pipes was detected before harvest. The red spores of Haematococcus pluvialis were harvested, dried into algal powder, and the dry weight and astaxanthin content of the algal powder were detected. The results are shown in Table 2. As can be seen from Table 2, the inhibitory effect of adding caspofungin or β-mannanase alone on chytrid fungus was significantly weaker than that of the two combined. Moreover, the dry weight and astaxanthin content of the algal powder harvested in the single-use group were different from those in the normal control group.

[0047] Table 2 Comparison of the effects of adding caspofungin and β-mannanase on green algae cultivation

[0048]

[0049] Compared with the blank control group, **P<0.01, ***P<0.001; compared with the normal control group, ##P<0.01, ###P<0.001

[0050] Example 3 (Green Algae Cultivation Stage)

[0051] The method for preventing contamination of Haematococcus pluvialis in the green algae stage and Chytrid fungi in PB bags includes the following steps:

[0052] (1) Add Haematococcus pluvialis green algae solution and BG11 culture medium to an outdoor PB bag, then add filtered water to the calibration volume, so that the Haematococcus pluvialis green motile cells grow at a rate of 2×10⁻⁶. 4 The cells were cultured at a density of approximately 3-5 × 10⁶ cells / ml the next day, and the number of green motile cells was approximately 3-5 × 10⁶. 4 Using PB bags containing 100 cells / ml as the experimental subject, and 100 ml of bags containing only red, immobile cells at a density of 100 × 10⁻⁶ cells / ml, the experiment was conducted. 4 Haematococcus pluvialis infection solution with a concentration of 1 / ml and an infection rate of 50% was added to the PB bags of each experimental group. At this time, the total infection rate of Haematococcus pluvialis chytrid fungus in the PB bags of the experimental groups was about 0.6%-1.0%. The algal solution without chytrid fungus infection was used as the normal control group. (2) 2ppm caspofungin + 3ppm β-mannanase were added to the PB bags of each experimental group. At the same time, the control group without adding any reagents to control chytrid fungus was used as the blank control group. (3) The chytrid fungus contamination, number of motile cells and number of dividing cells were monitored daily. (4) The pH value of the algal solution in the PB bags was detected daily. The group with a pH value less than 8.0 was adjusted to an aeration ratio of carbon dioxide: air = 2:500 (v / v). The constant pH value was always below 8.0. The group with a pH value maintained between 8.0 and 9.5 had an aeration ratio of For example, the ratio of carbon dioxide to air was controlled at 2:125 (v / v), and the pH value was adjusted to 2:10 (v / v) for groups with a ventilation ratio greater than 9.5. (5) The algal solution infected with chytrid fungi was added as day 0, followed by 6 days of culture. The entire process was carried out in PB bags for a total of 8 days. The chytrid fungi infection rate, number of motile cells and number of dividing cells in the green spore cells of PB bags were detected on the last day. (6) The algal solution in 6 PB bags of the same experimental group was then connected to a GP pipeline with a volume of 3000L for culture. Each experimental group had three GP pipelines. After 20 days of culture, the chytrid fungi infection rate of red spore cells in the GP pipeline was detected before harvest. The red spores of Haematococcus pluvialis were harvested and dried into algal powder. The dry weight of the algal powder and the content of astaxanthin were then detected. The test results are shown in Table 3. Table 3 shows that the infection rate of Chytridium in the group with pH maintained between 8.0 and 9.5 after adding caspofungin and β-mannanase was significantly lower than that in other groups. In addition, the number of motile cells and dividing cells in the PB bag was significantly higher than that in other groups, and the dry weight and content of the final harvested algal powder were also significantly higher than those in other groups.

[0053] Table 3 Comparison of the effects of pH value on green algae cultivation

[0054]

[0055] Compared with the group with pH ≤ 9.5, *P < 0.05, **P < 0.01

[0056] Example 4 (Red Algae Cultivation Stage)

[0057] The method for preventing Haematococcus pluvialis red algae stage chytrid fungus contamination in GP pipelines includes the following steps:

[0058] (1) Haematococcus pluvialis at 2×10 4 After culturing at a density of cells / ml in outdoor GP tubing for three days, the cells were subjected to stress culture with red-conversion medium to transform all green motile cells into red akinetic spore cells. The next day, the number of akinetic spore cells was approximately 5 × 10⁶. 4 GP tubing with cells / ml was used as the experimental tubing, containing 500 ml of red immobile cells at a density of 100 × 10⁻⁶ cells. 4 Haematococcus pluvialis infection solution with 50% infection rate was added to the pipes of each experimental group. At this time, the total infection rate of Haematococcus pluvialis chytrids in the pipes of the experimental group was about 0.1%-0.2%; (2) Caspofungin and β-mannanase of different concentrations were added to the pipes of each experimental group, and the control group without adding any reagents was used as a blank control group; (3) The chytrid contamination was monitored daily; (4) The pH value of the algal solution in the GP pipe was detected daily, and the carbon dioxide and air ventilation were adjusted to ensure that the pH value was constant between 8.0 and 9.5. (5) The day of salt addition was taken as day 0. After 12 days of cultivation, the infection rate was detected by sampling, and Haematococcus pluvialis spores were harvested and dried into algal powder. The infection rate of chytrids, the dry weight of algal powder and the content of astaxanthin were detected. The detection results are shown in Table 4. Table 4 shows that the infection rate of each experimental group with added caspofungin and β-mannanase was significantly lower than that of the blank control group, and the dry weight and astaxanthin content were significantly higher than those of the blank control group. Among them, the infection rate, algal powder dry weight and astaxanthin content of the group with added 2 ppm caspofungin + 3 ppm β-mannanase were significantly different from those of the blank control group.

[0059] Table 4 Comparison of the effects of caspofungin and β-mannanase concentrations on red algae cultivation

[0060]

[0061] Compared with the blank control group, **P<0.01, ***P<0.001

[0062] Microscopic examination was performed on the inhibition of Chytridium in the GP tubing, as shown in the attached figure. Figure 2 From the appendix Figure 2It can be seen that both the blank control group and the drug-treated group had Chytridella infection rates on day 0, but the degree of infection was not significantly different. On day 12 of culture, all cells in the blank control group died, and there was no significant difference in the number of red spore cells between the normal control group and the drug-treated group. This indicates that the drug treatment of the present invention effectively inhibited Chytridella infection.

[0063] Example 5 (Red Algae Cultivation Stage)

[0064] The method for preventing Haematococcus pluvialis red algae stage chytrid fungus contamination in GP pipelines includes the following steps:

[0065] (1) Haematococcus pluvialis at 2×10 4 After culturing at a density of cells / ml for three days in 3000L GP tubing in an outdoor environment, the cells were subjected to stress culture with red-conversion medium to completely transform the green motile cells into red akinetic spore cells. The next day, the number of akinetic spore cells was approximately 5 × 10⁶. 4 GP tubing with cells / ml was used as the experimental tubing, containing 500 ml of red immobile cells at a density of 100 × 10⁻⁶ cells. 4 Haematococcus pluvialis infection solution with a concentration of 1 / ml and an infection rate of 50% was added to the pipes of each experimental group. At this time, the total infection rate of Haematococcus pluvialis chytrids in the pipes of the experimental group was about 0.1%-0.2%. The normal control group was not added with the infected algae solution. (2) Then, 2ppm caspofungin, 5ppm caspofungin, 3ppm β-mannanase, 5ppm β-mannanase, 2ppm caspofungin + 3ppm β-mannanase were added to the pipes of each experimental group with the added infected algae solution. At the same time, the blank control group was controlled without any reagents. (3) The chytrid contamination was monitored daily. (4) The pH value of the algae solution in the GP pipe was detected daily, and the carbon dioxide and air ventilation were adjusted to ensure that the pH value was constant between 8.0 and 9.5. (5) The day of salt addition was taken as day 0. After 12 days of cultivation, the infection rate was detected by sampling, and Haematococcus pluvialis spores were harvested and dried into algae powder. The dry weight of algae powder and the content of astaxanthin were detected. The detection results are shown in Table 5. The inhibition rate of Chytridium was significantly higher when caspofungin or β-mannanase was added alone compared with the blank control group. The inhibition rate of the combined use of the two was significantly different from that of the blank control group. There was no significant difference in the dry weight of algal powder and the content of astaxanthin between the combined use group and the uninfected normal group.

[0066] Table 5 Comparison of the effects of caspofungin and β-mannanase alone and in combination on red algae cultivation

[0067]

[0068] Compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001; compared with the uninfected normal control group, #P<0.05, ##P<0.01, ###P<0.001

[0069] The combined use of caspofungin and β-mannanase provides a more thorough inhibition of Chytridaconia, effectively reducing the impact of Chytridaconia on Haematococcus pluvialis cultivation.

[0070] Example 6 (Red Algae Cultivation Stage)

[0071] The method for preventing Haematococcus pluvialis red algae stage chytrid fungus contamination in GP pipelines includes the following steps:

[0072] (1) Haematococcus pluvialis at 2×10 4 After culturing at a density of cells / ml for three days in 3000L GP tubing in an outdoor environment, the cells were subjected to stress culture with red-conversion medium to completely transform the green motile cells into red akinetic spore cells. The next day, the number of akinetic spore cells was approximately 5 × 10⁶. 4 GP tubing with cells / ml was used as the experimental tubing, containing 500 ml of red immobile cells at a density of 100 × 10⁻⁶ cells. 4 Haematococcus pluvialis infection solution with a concentration of 1 / ml and an infection rate of 50% was added to the pipes of each experimental group. At this time, the total infection rate of Haematococcus pluvialis chytrids in the pipes of the experimental groups was about 0.1%-0.2%; (2) Then, 2ppm caspofungin + 3ppm β-mannanase were added to the pipes; (3) The chytrid contamination was monitored daily; (4) The pH value of the algal solution in the GP pipes was measured daily. For groups with a pH value less than 8.0, the ventilation ratio was adjusted to carbon dioxide: air = 2:500 (L / h) and kept constant. The pH value was always below 8.0; the group with pH value maintained between 8.0 and 9.5 had the ventilation ratio controlled at carbon dioxide: air = 2:125 (L / h), and the group with pH value greater than 9.5 had the ventilation ratio adjusted to carbon dioxide: air = 2:10 (L / h); (5) the day of salt addition was taken as day 0, and after 12 days of cultivation, samples were taken to detect the infection rate, and Haematococcus pluvialis spores were harvested and dried into algal powder. The dry weight of algal powder and astaxanthin content were detected. The infection rate, dry weight of algal powder and astaxanthin content are shown in Table 6. The inhibition rate of chytrids was significantly higher when caspofungin or β-mannanase was added alone than that of the blank control group. The inhibition rate of the two combined was significantly different from that of the blank control group, and there was no significant difference in the dry weight of algal powder and astaxanthin content between the combined group and the uninfected normal group.

[0073] Table 6 Comparison of the effects of pH value on red algae cultivation

[0074] Infection rate (%) 6.67±0.06** 0.23±0.06 4.33±1.53** Dry weight (g / L) 1.01±0.02* 1.46±0.04 1.12±0.04* Astaxanthin content (%) 4.08±0.04* 4.94±0.06 4.36±0.03*

[0075] Compared with the group with pH ≤ 9.5, *P < 0.05, **P < 0.01

[0076] The combined use of caspofungin and β-mannanase showed the best inhibitory effect on Chrystridium typhimurium at a pH of 8.0-9.5. It still had a good inhibitory effect at pH values ​​less than 8 or greater than 9.5, but the inhibition rate of Chrystridium typhimurium decreased.

[0077] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method of inhibiting chitin infection in Haematococcus cultivation, characterized in that, Caspofungin and β-mannanase were added to the algal solution of Haematococcus pluvialis infected with Chytridium; the concentration of caspofungin in the algal solution was 1-5 ppm, and the concentration of β-mannanase in the algal solution was 1-6 ppm.

2. The method of inhibiting chitin fungi infection in Haematococcus cultivation according to claim 1, characterized in that, The concentration ratio of caspofungin to β-mannanase is 1:1-2.

3. The method for inhibiting chytrid fungal infection in Haematococcus pluvialis cultivation according to claim 1, characterized in that, Control the pH of the algae solution to 8.0-9.

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4. The method for inhibiting chytrid fungal infection in Haematococcus pluvialis cultivation according to claim 3, characterized in that, The pH value of the algal solution is controlled by adjusting the ratio of carbon dioxide and air introduced into the solution.

5. The method for inhibiting chytrid fungal infection in Haematococcus pluvialis cultivation according to claim 1, characterized in that, The Haematococcus pluvialis mentioned are Haematococcus pluvialis green algae cultured in outdoor PB bags or Haematococcus pluvialis red algae cultured in GP pipes.

6. The application of caspofungin and β-mannanase in the combined use to inhibit chytrid fungi during the cultivation of Haematococcus pluvialis, characterized in that... The concentration of caspofungin in the algal solution is 1-5 ppm, and the concentration of β-mannanase in the algal solution is 1-6 ppm.

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