Prevention and control method for protozoa and chytrid outbreak in haematococcus pluvialis culture process
By controlling the temperature during the deep-water dynamic closed breeding of Rainy Chronicus, the outbreak of protozoa and chytrids was reduced, and the problems of algae cell production reduction and economic losses during the breeding process were solved, and efficient and economical astaxanthin production and low-risk breeding effects were achieved.
Patent Information
- Application Number
- CN202411948749.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-03-28
AI Technical Summary
During the dynamic and closed breeding of Rainy Chronicus in deep water, the outbreak of protozoa and chytrids leads to reduced production or extinction of algae cells, causing economic losses and forming a vicious cycle.
By controlling the breeding temperature, the temperature is lowered to inhibit the outbreak of protozoa and chytrids. Specific methods include reducing the breeding temperature by 5-7°C in the green and red cultivation stages, and combining deep water dynamic closed aquaculture equipment and dual-stage reverse osmosis treatment, and immersion and disinfection of sodium hypochlorite solution.
It effectively inhibits the outbreak of protozoa and chytrids, increases astaxanthin production, reduces breeding risks, ensures 50% to 100% harvest, and reduces costs.
Smart Images
Figure CN119709414A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microalgae biotechnology, and in particular relates to a method for preventing and controlling protozoa and chytrid outbreaks during the deep-water dynamic closed cultivation of Haematococcus pluvialis. Background Art
[0002] Haematococcus pluvialis is a single-celled freshwater green algae that, under specific conditions, can accumulate astaxanthin at levels of 1% to 5% of its dry weight. It is widely used in food, cosmetics, aquaculture, and other fields. It is recognized as the best natural biological resource for producing natural astaxanthin and holds excellent development prospects.
[0003] However, during large-scale cultivation of Haematococcus pluvialis, regardless of whether the aquaculture system is connected to the outside world, it is inevitably exposed to pollutants (such as protozoa, chytrid fungi, and other algae) during algae seed inoculation, nutrient addition, and aeration. This can contaminate the aquaculture system, leading to reduced or even complete crop failure. Furthermore, pollutants remaining in the aquaculture system can become a source of contamination in subsequent Haematococcus pluvialis cultivation processes, creating a vicious cycle.
[0004] Among them, protozoa pose a particularly serious threat to Haematococcus pluvialis by feeding on it. For example, rotifers, a type of protozoan, can consume over 500 algal cells per hour, completely devouring them within a few days. Furthermore, rotifer predation on microalgae can further contaminate other algae and promote the proliferation of bacteria and other microorganisms, resulting in significant economic losses.
[0005] Chytrid is an obligate parasitic pathogen of Haematococcus pluvialis. During the large-scale cultivation process of Haematococcus pluvialis, the spores are infected by the chytrid in the green algae stage, which will lead to a large outbreak of chytrid infection when entering the deep-water dynamic aquaculture equipment for forced cultivation in the later stage. Once the infection rate of the chytrid exceeds 50% for the Haematococcus pluvialis cultured in the deep-water dynamic aquaculture equipment, all algae cells will die within 2 to 3 days, resulting in the large amount of manpower, material resources and algae species invested in the early cultivation process to obtain no harvest, which is a heavy loss to the enterprise.
[0006] At present, the control of protozoa and chytrid fungus contamination in large-scale cultivation of Haematococcus pluvialis is mainly based on prevention, while a combination of physical and chemical methods is used for prevention and control.
[0007] Common physical methods for water treatment include high temperature and high pressure sterilization for water in laboratories or small-scale aquaculture systems, and two-stage reverse osmosis treatment for water in medium or large-scale aquaculture.
[0008] Chemical methods are commonly used to treat aquaculture equipment, such as soaking and disinfecting Haematococcus pluvialis aquaculture equipment with 100-200 ppm sodium hypochlorite, but a large amount of water is needed to remove the residual chlorine in the equipment, or 10% sodium thiosulfate is added for neutralization.
[0009] The above methods can only ensure that the water and equipment are pollution-free in the initial state, but pollution problems will still occur during the aquaculture operation process due to human operation or environmental reasons, and the costs of both treatment methods are extremely high.
[0010] Currently, the methods for controlling biological contamination in the aquaculture process include adding additional chemical reagents and surfactants. However, these control methods have the problems of obvious damage or even death of algal cells, rapid recurrence of contamination, residual additional chemical components, high treatment costs, and unsuitability for large-scale use. Summary of the Invention
[0011] The technical problem addressed by this invention is to provide a method for preventing and controlling protozoan and chytrid outbreaks during the deepwater, dynamic, and closed aquaculture of Haematococcus pluvialis. By lowering the temperature, protozoan and chytrid outbreaks can be effectively suppressed, reducing the low astaxanthin content and cell death in Haematococcus pluvialis caused by protozoan and chytrid infections, lowering aquaculture risks, and increasing astaxanthin production after infection. This method significantly inhibits protozoan and chytrid outbreaks during both the green and red aquaculture stages. Specifically, by controlling the inlet water temperature, feed temperature, and aquaculture temperature, this method can prevent protozoan and chytrid outbreaks throughout the aquaculture cycle, achieving a harvest rate of 50% to 100%.
[0012] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0013] Methods for preventing and controlling protozoan and chytrid outbreaks during Haematococcus pluvialis cultivation include the following steps:
[0014] 1) Using deep-water dynamic closed aquaculture equipment, the water body is treated by double-stage reverse osmosis;
[0015] 2) Soak the aquaculture equipment in sodium hypochlorite solution for disinfection; after disinfection, rinse the aquaculture equipment with pure water until the residual chlorine content in the rinse water is less than 0.5 mg / L;
[0016] 3) First, water is poured into the culture equipment, and then the BBM culture medium stock solution that has been sterilized at high temperature and high pressure and cooled is added;
[0017] 4) Adding Haematococcus pluvialis liquid and then pure water for green culture and red culture;
[0018] The cultivation temperature of the green culture stage of Haematococcus pluvialis is 21±1°C or the cultivation temperature is 5-6°C lower than the general green culture temperature of 25-28°C;
[0019] The culture temperature of the Haematococcus pluvialis in the red culture stage is 24±1° C., or the culture temperature is 5-7° C. lower than the general red culture temperature of 28-32° C.
[0020] Preferably, in step 1), the specific method and operating conditions for the double-stage reverse osmosis treatment of the water body are: the first stage reverse osmosis removes large particle impurities; the second stage reverse osmosis further removes residual dissolved solids and ions.
[0021] Preferably, in step 2), the concentration of the sodium hypochlorite solution is 150-200 ppm.
[0022] Preferably, in step 2), the soaking and disinfection time is ≥ 20 h.
[0023] Preferably, in step 3), the specific operating conditions of the high temperature and high pressure sterilization are: 121° C., pressure of 0.1-0.3 MPa, and sterilization for 15 minutes.
[0024] Preferably, in step 3), the temperature of the injected water is 18-22°C; the temperature of the product after high temperature and high pressure sterilization and cooling is 18-22°C.
[0025] Preferably, in step 4), the temperature of the Haematococcus pluvialis liquid in the green culture stage is 21±1°C, and the temperature in the red culture stage is 24±1°C.
[0026] Unless otherwise specified, all components and materials in the present invention can be purchased commercially, and the equipment used in the present invention can adopt conventional equipment in the relevant field or refer to the existing technology in the relevant field.
[0027] Compared with the prior art, the present invention has the following beneficial effects :
[0028] 1) The control method of the present invention is low-cost and guarantees economic returns. While both physical and chemical treatments incur significant additional costs, temperature control can effectively control protozoa and chytrid fungi without requiring additional equipment, significantly reducing costs. Immediately implementing cooling measures upon detection of large numbers of protozoa and chytrid fungi allows for more time for harvesting, minimizing economic losses for the enterprise and potentially achieving a 50%-100% yield.
[0029] 2) This method is simple to operate, highly feasible, and does not cause water pollution or damage to equipment. It uses temperature control and does not add any chemical reagents, causing no harm to Haematococcus pluvialis, producing no toxic substances, and ensuring that the harvested algae powder contains no unnecessary added chemicals. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.
[0031] Figure 1 The microscopic examination of algal cells in the control group and experimental group of Haematococcus pluvialis during the green culture stage in Example 1;
[0032] Figure 2 These are microscopic examination images of algal cells in the control and experimental groups of Haematococcus pluvialis during the red culture stage in Example 1. DETAILED DESCRIPTION
[0033] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.
[0034] It should be noted that when an element is referred to as being “fixed on” or “disposed on” another element, it may be directly on the other element or indirectly on the other element. When an element is referred to as being “connected to” another element, it may be directly connected to the other element or indirectly connected to the other element.
[0035] It should be understood that the terms "length", "width", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limiting the present invention.
[0036] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature identified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.
[0037] In the present invention, the "Haematococcus pluvialis" is publicly available on the market and is not particularly limited. The "Haematococcus pluvialis" used in this application can be selected, for example, from the algae species ZMB22 (Eukaryota; Viridiplantae; Chlorophyta; core chlorophytes; Chlorophyceae; CS clade; Chlamydomonadales; Haematococcaceae; Haematococcus.) screened and published by Guizhou Zhongming Biotechnology Co., Ltd. Alternatively, algae species such as Haematococcus pluvialis NIES-144 (GenBank: AB360747.1) isolated from Japan (Nakada et al. 2008) can be selected. These algae species have extremely high similarity (E value 0.0, per.Ident 100%, Acc.len 1728).
[0038] As one aspect of the present invention, a method for preventing and controlling protozoan and chytrid outbreaks during Haematococcus pluvialis cultivation comprises the following steps:
[0039] 1) Using deep-water dynamic closed aquaculture equipment, the water body is treated by double-stage reverse osmosis;
[0040] 2) Soak the aquaculture equipment in sodium hypochlorite solution for disinfection; after disinfection, rinse the aquaculture equipment with pure water until the residual chlorine content in the rinse water is less than 0.5 mg / L;
[0041] 3) First, water is poured into the culture equipment, and then the BBM culture medium stock solution that has been sterilized at high temperature and high pressure and cooled is added;
[0042] 4) Adding Haematococcus pluvialis liquid and then pure water for green culture and red culture;
[0043] The cultivation temperature of the green culture stage of Haematococcus pluvialis is 21±1°C or the cultivation temperature is 5-6°C lower than the general green culture temperature of 25-28°C;
[0044] The culture temperature of the Haematococcus pluvialis in the red culture stage is 24±1° C., or the culture temperature is 5-7° C. lower than the general red culture temperature of 28-32° C.
[0045] It can be understood that the current common cultivation temperature for the green stage of Haematococcus pluvialis is 25-28°C, and the cultivation temperature for the red stage of Haematococcus pluvialis is 28-32°C; therefore, the prevention and control means of this application is a mode of cooling cultivation at the existing common cultivation temperature.
[0046] According to certain embodiments of the present invention, in step 1), the water body is treated by a two-stage reverse osmosis device, which is an existing device. The working principle is mainly based on reverse osmosis technology, which is a process of using pressure difference to make water pass through a semi-permeable membrane. In this way, impurities, bacteria, viruses and other substances in the water are filtered out, thereby obtaining pure water; specifically, the two-stage reverse osmosis system includes two main parts: a primary reverse osmosis and a secondary reverse osmosis; a primary reverse osmosis: tap water first passes through a pretreatment system to remove large particles of impurities, and then enters the primary reverse osmosis system; at this stage, water molecules are forced to pass through the reverse osmosis membrane by high pressure, while dissolved solids, microorganisms and some dissolved ions are retained outside the membrane, thereby obtaining relatively pure water; a secondary reverse osmosis: the pure water produced by the primary reverse osmosis then enters the secondary reverse osmosis system for treatment; this stage further removes residual dissolved solids and ions to provide water of higher purity.
[0047] According to certain embodiments of the present invention, in step 2), the concentration of the sodium hypochlorite solution is 150-220 ppm.
[0048] According to some embodiments of the present invention, in step 2), the immersion disinfection time is ≥≥20 h.
[0049] According to certain embodiments of the present invention, in step 3), the specific operating conditions of the high temperature and high pressure sterilization are: 121° C., pressure of 0.1-0.3 MPa, and sterilization for 15 minutes.
[0050] According to certain embodiments of the present invention, in step 3), the temperature of the injected water is 18-22°C; and the temperature to which the product is sterilized at high temperature and high pressure and then cooled is 18-22°C.
[0051] According to some embodiments of the present invention, in step 4), the temperature of the Haematococcus pluvialis liquid in the green culture stage is 21±1°C, and the temperature in the red culture stage is 24±1°C.
[0052] Example 1
[0053] The method for preventing and controlling protozoan and chytrid outbreaks during Haematococcus pluvialis cultivation includes the following steps:
[0054] 1) Use deep water dynamic closed aquaculture equipment with a liquid holding capacity of 6m 3 , the water is treated by double-stage reverse osmosis;
[0055] 2) Soak the aquaculture equipment in 200ppm sodium hypochlorite solution for 24 hours; after disinfection, rinse the aquaculture equipment with pure water until the residual chlorine content in the rinse water is less than 0.5mg / L;
[0056] 3) First, fill water into the aquaculture equipment to 4m 3Fill the tube with water at a temperature of 18-22°C, then add the BBM medium stock solution that has been sterilized at high temperature and high pressure and cooled to 22°C;
[0057] 4) Add 20-22℃ Haematococcus pluvialis liquid according to the inoculation amount of 10%, and then add pure water to the specified 5.5ml 3 Green culture begins at the scale line: the culture temperature of the green culture stage of the Haematococcus pluvialis is 21±1°C, and the culture is carried out for 9 days; samples are taken daily to examine the state of the algal cells under a microscope, and the algal abundance and biomass are recorded. There is no outbreak of protozoa and chytrid fungi, and the algal cells grow normally. This group is used as the experimental group.
[0058] See Table 1 below for a comparison of the effects of temperature on algal abundance and biomass of Haematococcus pluvialis during the green culture stage.
[0059] Comparative Example 1
[0060] A method for cultivating Haematococcus pluvialis in a closed deep-water dynamic cultivation device comprises the following steps:
[0061] 1) Use deep water dynamic closed aquaculture equipment with a liquid holding capacity of 6m 3 , the water is treated by double-stage reverse osmosis;
[0062] 2) Soak the aquaculture equipment in 200ppm sodium hypochlorite solution for 24 hours; after disinfection, rinse the aquaculture equipment with pure water until the residual chlorine content in the rinse water is less than 0.5mg / L;
[0063] 3) First, fill water into the aquaculture equipment to 4m 3 Fill the tube with water at a temperature of 18-22°C, then add the BBM medium stock solution that has been sterilized at high temperature and high pressure and cooled to 22°C;
[0064] 4) Add 20-22℃ Haematococcus pluvialis liquid according to the inoculation amount of 10%, and then add pure water to the specified 5.5ml 3 Green culture begins at the scale line: the culture temperature of the green culture stage of the Haematococcus pluvialis is 26±1°C, and the culture is carried out for 9 days; samples are taken daily for microscopic examination of the algal cell status, and the algal abundance and biomass are recorded. On the 3rd to 4th day of culture, a large number of protozoa and chytrid fungi are found to have broken out, and the number of algal cells has dropped sharply until the algal cells are almost extinct. This group serves as the control group.
[0065] The difference between this comparative example and Example 1 is that: in step 4), the cultivation temperature in the green cultivation stage of this comparative example is 26±1°C; the cultivation temperature in the green cultivation stage of Example 1 is 21±1°C.
[0066] Table 1: Comparison of the effects of green culture temperature on algal abundance and biomass of Haematococcus pluvialis during green culture
[0067]
[0068]
[0069] Figure 1 The microscopic examination pictures of the algae cells of the control group and the experimental group of Haematococcus pluvialis in the green culture stage in Comparative Example 1 and Example 1;
[0070] Thus, it can be seen from Table 1 that the experimental group of Example 1 was able to control protozoa and chytrid by lowering the green culture temperature, preventing their outbreak and allowing the Haematococcus pluvialis cells to grow normally; while the control group of Comparative Example 1 had an excessively high green culture temperature, resulting in an outbreak of protozoa and chytrid, leading to the rapid extinction of the Haematococcus pluvialis cells.
[0071] Example 2
[0072] The method for preventing and controlling protozoan and chytrid outbreaks during Haematococcus pluvialis cultivation includes the following steps:
[0073] 1) Use deep water dynamic closed aquaculture equipment with a liquid holding capacity of 6m 3 , the water is treated by double-stage reverse osmosis;
[0074] 2) Soak the aquaculture equipment in 200ppm sodium hypochlorite solution for 24 hours; after disinfection, rinse the aquaculture equipment with pure water until the residual chlorine content in the rinse water is less than 0.5mg / L;
[0075] 3) First, fill water into the aquaculture equipment to 4m 3 Fill the tube with water at a temperature of 18-22°C, then add the BBM medium stock solution that has been sterilized at high temperature and high pressure and cooled to 22°C;
[0076] 4) Add 20-22℃ Haematococcus pluvialis liquid according to the inoculation amount of 10%, and then add pure water to the specified 5.5ml 3 Green culture begins at the scale line: the culture temperature of the green culture stage of the Haematococcus pluvialis is 21±1°C, and the culture is carried out for 9 days; samples are taken daily to examine the algal cell status, and the algal abundance and biomass are recorded. There is no outbreak of protozoa and chytrid fungi, and the algal cells grow normally;
[0077] 5) The normally grown Haematococcus pluvialis was then cultured in the red culture stage at a temperature of 24 ± 1°C. Samples were taken daily for microscopic examination of algal cell status, and biomass and astaxanthin content were recorded. The algae were cultured for a total of 7 days. If no protozoa or chytrid outbreaks occurred, the algae were harvested after the culture period ended. This group served as the experimental group.
[0078] See Table 2 below for a comparison of the effects of red culture temperature on biomass and astaxanthin of Haematococcus pluvialis during the red culture stage.
[0079] Comparative Example 2
[0080] A method for cultivating Haematococcus pluvialis in a closed deep-water dynamic cultivation device comprises the following steps:
[0081] 1) Use deep water dynamic closed aquaculture equipment with a liquid holding capacity of 6m 3 , the water is treated by double-stage reverse osmosis;
[0082] 2) Soak the aquaculture equipment in 200ppm sodium hypochlorite solution for 24 hours; after disinfection, rinse the aquaculture equipment with pure water until the residual chlorine content in the rinse water is less than 0.5mg / L;
[0083] 3) First, fill water into the aquaculture equipment to 4m 3 Fill the tube with water at a temperature of 18-22°C, then add the BBM medium stock solution that has been sterilized at high temperature and high pressure and cooled to 22°C;
[0084] 4) Add 20-22℃ Haematococcus pluvialis liquid according to the inoculation amount of 10%, and then add pure water to the specified 5.5ml 3 Green culture begins at the scale line: the culture temperature of the green culture stage of the Haematococcus pluvialis is 21±1°C, and the culture is carried out for 9 days; samples are taken daily to examine the algal cell status, and the algal abundance and biomass are recorded. There is no outbreak of protozoa and chytrid fungi, and the algal cells grow normally;
[0085] 5) The normally grown Haematococcus pluvialis was then cultured in the red culture stage at a temperature of 30±1°C. Samples were taken daily for microscopic examination of algal cell status, and biomass and astaxanthin content were recorded. A large outbreak of protozoa and chytrid fungi was observed on the second day of culture, and almost all algal cells were exterminated on the fourth day of culture. This group served as the control group.
[0086] Figure 2 These are microscopic examination images of algal cells in the control group and experimental group of Haematococcus pluvialis during the red culture stage in Comparative Example 2 and Example 2.
[0087] Table 2: Comparison of the effects of red culture temperature on biomass and astaxanthin of Haematococcus pluvialis during red culture
[0088]
[0089] Thus, it can be seen from Table 2 that the experimental group of Example 2 was able to control protozoa and chytrid by lowering the red culture temperature, preventing their outbreak and allowing algal cells to grow normally and accumulate astaxanthin; while the control group of Comparative Example 2 had an excessively high red culture temperature, resulting in an outbreak of protozoa and chytrid, leading to a rapid death of algal cells.
[0090] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications based on the above description are possible. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.
Claims
1. A method for preventing and controlling the outbreak of protozoa and chytrid fungi during the cultivation of Haematococcus pluvialis, characterized in that: The steps include: 1) Use deep-water dynamic closed aquaculture equipment, and the water is treated by double-stage reverse osmosis; 2) Soak the aquaculture equipment in sodium hypochlorite solution for disinfection; after disinfection, rinse the aquaculture equipment with pure water until the residual chlorine content in the rinse water is less than 0.5 mg / L; 3) First, water is injected into the culture equipment, and then the BBM culture medium mother solution that has been sterilized at high temperature and high pressure and cooled is added; 4) Adding Haematococcus pluvialis liquid and pure water for green culture and red culture; The cultivation temperature of the green cultivation stage of Haematococcus pluvialis is 21±1°C or the cultivation temperature is 5-6°C lower than the general green cultivation temperature of 25-28°C; The culture temperature of the red culture stage of Haematococcus pluvialis is 24±1°C or the culture temperature is 5-7°C lower than the general red culture temperature of 28-32°C.
2. The control method according to claim 1, characterized in that: In step 1), the water body is treated by two-stage reverse osmosis: the first stage reverse osmosis removes large particle impurities; the second stage reverse osmosis further removes residual dissolved solids and ions.
3. The control method according to claim 1, characterized in that: In step 2), the concentration of the sodium hypochlorite solution is 150-200 ppm.
4. The control method according to claim 1, characterized in that: In step 2), the immersion disinfection time is ≥ 20 hours.
5. The control method according to claim 1, characterized in that: In step 3), the specific operating conditions of the high temperature and high pressure sterilization are: temperature of 121° C., pressure of 0.1-0.3 MPa, and time of 15 min.
6. The control method according to claim 1, characterized in that: In step 3), the temperature of the injected water is 18-22°C; the temperature of the product after high temperature and high pressure sterilization and cooling is 18-22°C.
7. The control method according to claim 1, characterized in that: In step 4), the temperature of the Haematococcus pluvialis liquid in the green culture stage is 21±1°C, and the temperature in the red culture stage is 24±1°C.