Method for removing protozoa in chlorella species conservation bottle
By using sodium hypochlorite disinfection and sodium thiosulfate neutralization methods, the precipitate was separated and secondary disinfection was performed, which solved the problem of protozoa contamination in Chlorella culture medium and achieved an efficient and gentle removal effect.
Patent Information
- Application Number
- CN202510747694.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-05
- Publication Date
- 2025-10-10
AI Technical Summary
Existing technologies are difficult to effectively remove protozoan contamination from Chlorella culture medium, and commonly used chemicals have an impact on algae, so a gentle and effective removal method is needed.
Sodium hypochlorite is used to disinfect and separate the precipitate, and sodium thiosulfate is used to neutralize the residual chlorine, and a secondary disinfection treatment is performed to ensure the purity of the algae liquid.
It significantly improves the protozoa removal rate, ensures the purity and healthy growth of Chlorella culture solution, and reduces the impact of chemicals on algae.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of chlorella cultivation, and in particular to a method for removing protozoa in a chlorella seed bottle. Background Art
[0002] Chlorella vulgaris, a typical bait microalgae, is a single-celled microalgae belonging to the genus Chlorella in the family Chlorellaceae. Common species include over a dozen, including Chlorella pyrenoidosa, Chlorella marinea, Chlorella oculata, and Chlorella ovata. Chlorella is an important bait microalgae, rich in various nutrients and possessing significant economic value. It is widely used in aquaculture, food, and feed production. However, the large-scale cultivation of microalgae baits is prone to protozoan contamination due to unavoidable factors, such as improper operation, climate change, and poor water quality. Common protozoa include Euplotes sp., Chaetonotus, and Amoeba proteus. These pests grow and reproduce in algal culture medium. When their populations reach a certain density, they can affect algal growth. This contaminated algal culture medium is difficult to expand and can even lead to culture failure. Currently, it's difficult to completely prevent protozoan contamination and damage to Chlorella. To address this issue, many institutions have experimented with methods to control protozoa in Chlorella culture fluids, but only a few methods are effective. For example, lowering the pH of the algae culture to 3.0 can kill spiky-nosed parasites in golden algae and flat algae cultures. Another approach is to use 0.03% concentrated ammonia to inhibit or kill protozoa. Chemicals such as formaldehyde, ammonia, and hydrogen peroxide are also commonly used to address protozoan contamination. While these chemicals can inhibit or kill harmful organisms, they can also have some impact on the cultured microalgae. Therefore, a gentle and effective protozoan removal method is needed. Summary of the Invention
[0003] The present invention aims to provide a method for removing protozoa from a Chlorella seed bottle, which can effectively eliminate protozoa without affecting the cultivation of Chlorella and has technical creativity and advancement in disinfection.
[0004] The technical solution is as follows: A method for removing protozoa from a Chlorella seed bottle.
[0005] Disinfection: Use sodium hypochlorite containing 8% available chlorine at a concentration of 250 mg / L to disinfect the algae culture tools and seawater to eliminate protozoa in the equipment and raw materials.
[0006] Inoculation: immediately after adding nutrients in the preservation bottle, inoculate the algae to culture, the initial concentration of algae cell number in the algae is 15 million / ml; after inoculating the algae, add disinfectant to ensure that the effective chlorine concentration in the algae body reaches 5mg / L, and the water body is placed for 1 hour.
[0007] Pouring the algae liquid: pour the upper algae liquid into another sterilized and disinfected glass container, and discard the lower protozoa and precipitate in the original container;
[0008] Residual chlorine neutralization: add sodium thiosulfate to the transferred algae liquid, and use sodium thiosulfate at a concentration of 0.5mg / L to neutralize the residual effective chlorine;
[0009] 48 hours after the first treatment, add disinfectants containing effective chlorine such as sodium hypochlorite, bleaching powder or bleaching powder to disinfect, after the water body is placed, observe whether there is precipitation, if there is, the container needs to be replaced again, if not, it does not need to be replaced, then add sodium thiosulfate at the corresponding concentration;
[0010] After the second treatment, use a microscope to check for protozoan reproduction every day (if there are still protozoa at this time, disinfect again), confirm the state and purity of the algae, then add culture solution to the algae liquid to provide necessary nutrients, and continue to culture the algae,
[0011] After 5 to 6 days, when the color of chlorella in the culture solution changes from light green to dark green, the expansion of the seed can be met, and the harvesting stage is entered, the chlorella is fished out with a filter screen and cleaned and treated.
[0012] The disinfectant is any one or two of sodium hypochlorite, bleaching powder or bleaching powder.
[0013] The ratio of the nutrient solution is sodium nitrate 74.8mg / L, urea 22.5mg / L, sodium dihydrogen phosphate 4.4mg, and ferric citrate 3.9mg / L.
[0014] Beneficial effects:
[0015] The Japanese capsule prawn culture method has the advantages of short culture period, high yield per mu, and less disease, and has important practical application value and market prospect. DETAILED DESCRIPTION
[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The terms used in the specification of the application are for the purpose of describing specific embodiments only and are not intended to limit this application. The terms "including" and "having" and any variations thereof in the specification and claims of this application are intended to cover non-exclusive inclusions. The terms "first" and "second" in the specification and claims of this application are used to distinguish different objects, not to describe a specific order.
[0017] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.
[0018] Example 1 Preparation: Chlorella vulgaris was cultured in seed flasks in a laboratory in Zhanjiang, Guangdong Province. The primary measures were to remove protozoa from the algal broth and to disinfect and sterilize the culture tools. Bleach, sodium thiosulfate, and other reagents, as well as glassware (e.g., beakers, culture flasks), a microscope, a graduated cylinder, a pipette, and an electronic balance, were prepared in advance. Before conducting the experiment, the laboratory environment was clean to avoid external contamination and a suitable culture environment (e.g., temperature, light, etc.) was established.
[0019] ⑵ Disinfect algae culture tools: Use sodium hypochlorite containing 8% available chlorine at a concentration of 250 mg / L to disinfect glassware (such as beakers, culture bottles) and the required seawater to eliminate protozoa. After disinfection for 12 to 24 hours, use sodium thiosulfate at a concentration of 20 mg / L to neutralize the residual chlorine.
[0020] (3) Inoculate algae species for cultivation: Add various nutrients required for the growth of Chlorella into the culture medium according to the culture medium formula. The culture medium formula is sodium nitrate 74.8 mg / L, urea 22.5 mg / L, sodium dihydrogen phosphate 4.4 mg, ferric citrate 3.9 mg / L, and then inoculate algae species for cultivation. The initial concentration of algae cells in the algae species is 15 million / ml.
[0021] (4) Observe the seed bottles: Chlorella was cultured normally in the laboratory, and the reproduction of protozoa in the algae solution was observed under a microscope every day. On the second day of culture, protozoa were found in the seed bottles, and the number of algae cells decreased from 15 million / ml at the time of inoculation to 11.5 million / ml.
[0022] 5. Disinfection of the algae solution in the seed bottle: First, accurately weigh 200mg of bleaching powder on an electronic balance. Filter seawater through an 80-mesh silk bag to remove any residue. Then, add water to a final volume of 1000mL, resulting in a bleaching powder solution concentration of 200mg / L. Each seed bottle contains 400mL of algae solution to be treated. The required bleaching powder solution is calculated to be 12.5mL and the required seawater is 87.5mL. Use a pipette to pipette a predetermined amount of bleaching powder solution into the algae solution at the desired concentration. Simultaneously, use a pipette to pipette a predetermined amount of seawater into the algae solution, ensuring that each bottle contains 500mL of liquid. The resulting mixture should have an available chlorine concentration of 5mg / L. Allow the treated water to stand for 1 hour without agitation to allow the protozoa to settle.
[0023] (6) Separation and Sedimentation: After one hour of stagnant sedimentation, observe the seed bottle and observe if a clear sediment has formed. Use a pipette to slowly pour the upper layer of algae solution into another sterilized conical flask. Pour slowly and steadily to avoid creating bubbles and disturbing the protozoa and sediment at the bottom. (If the sediment is accidentally disturbed, allow it to settle again before proceeding.) After the algae solution is separated, carefully discard the sediment and protozoa in the original container to ensure that no secondary contamination occurs during the process.
[0024] (7) Neutralization: After separation, the chlorine in the algae solution has a relatively small impact on the Chlorella, but it still has some effect. Therefore, we add sodium thiosulfate to neutralize the chlorine. After separation, we control the volume of the algae solution in each bottle to 400ml. The amount of sodium thiosulfate should be one-tenth of the chlorine content. Accurately weigh 0.2mg of sodium thiosulfate on an electronic balance and add it to the algae solution. Gently stir to ensure it is fully dissolved and evenly distributed. After adding the sodium thiosulfate, let it sit for 10-15 minutes to ensure that the available chlorine is completely neutralized.
[0025] ⑻ Repeat treatment: After neutralization, let the algae solution stand for 48 hours and observe the growth of the algae. Add bleaching powder in proportion to separate and precipitate, and then add sodium thiosulfate in the corresponding proportion to neutralize the solution to ensure the purity of the algae.
[0026] (9) Microscopic Observation and Cultivation: After a series of treatments, new culture medium (nutrient solution formula: 74.8 mg / L sodium nitrate, 22.5 mg / L urea, 4.4 mg sodium dihydrogen phosphate, 3.9 mg / L ferric citrate) was added to the algae solution to provide the necessary nutrients. After two days of cultivation, the treated algae solution was observed under a microscope to confirm that the algae cell morphology and number were normal, that there were no residual protozoa, and that the algal cell count reached 30 million / ml. The final observation results were very positive, indicating that the Chlorella culture was successful.
[0027] Experimental purpose: To compare the inactivation rates of protozoa removal methods in Chlorella seed bottles using single and secondary treatments, and to verify the effectiveness and advantages of secondary treatment.
[0028] 2. Experimental Materials and Methods
[0029] Experimental materials: prepare multiple seed bottles of the same specifications, collect Chlorella algae liquid containing protozoa, prepare sodium hypochlorite containing 8% effective chlorine, sodium thiosulfate, bleaching powder and other reagents, as well as glassware, microscope, measuring cylinder, pipette, electronic balance and other experimental equipment.
[0030] Experimental Method: The collected algae liquid was evenly distributed into multiple seed bottles, designated Group A (single treatment group) and Group B (secondary treatment group). Following the methods described in the Summary of the Invention, the algae culture tools and seawater in both groups of seed bottles were disinfected and inoculated with the same starting concentration of algae (15 million micrograms / ml). Group A was sterilized, allowed to stand, and then the algae liquid was poured out and the residual chlorine neutralized. No secondary disinfection was performed; Group B followed the full secondary disinfection process.
[0031] 3. Experimental data recording and analysis
[0032] Protozoa counting method: Equal amounts of algal fluid samples were collected daily before treatment, after a single treatment (Group A), and after a second treatment (Group B). Protozoa were observed and counted using a microscope. To ensure data accuracy, multiple fields of view were observed for each sample, and the average protozoa count was used as the average.
[0033] Experimental data
[0034]
[0035] Inactivation rate calculation: The inactivation rate calculation formula is: Inactivation rate (%) = (number of protozoa before treatment - number of protozoa after treatment) ÷ number of protozoa before treatment × 100%. According to this formula, the inactivation rate of Group A after a single treatment was: (800-200) ÷ 800 × 100% = 75%; the inactivation rate of Group B after a double treatment was: (800-50) ÷ 800 × 100% = 93.75%.
[0036] 4. Experimental Conclusion: Comparison of experimental data shows that the inactivation rate of the secondary treatment (93.75%) is significantly higher than that of the single treatment (75%). This indicates that the secondary treatment of protozoa in Chlorella vials can more effectively reduce the number of protozoa and improve the purity of the Chlorella culture environment. This further verifies the importance and effectiveness of the secondary treatment step in the protozoa removal process and strongly supports the practical application of this removal method.
[0037] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all the embodiments. They provide preferred embodiments of the present application, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application has been described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions described in the aforementioned specific embodiments, or to make equivalent replacements for some of the technical features therein. Any equivalent structure made using the contents of the present application specification, directly or indirectly used in other related technical fields, is also within the scope of patent protection of the present application.
Claims
1. A method for removing protozoa from a Chlorella seed bottle, characterized by: Disinfection: Use sodium hypochlorite containing 8% available chlorine at a concentration of 250 mg / L to disinfect the algae culture tools and seawater to eliminate protozoa in the equipment and raw materials. Inoculation: After adding nutrients to the seed bottle, immediately inoculate the algae seed for cultivation. The initial concentration of algae cells in the algae seed is 15 million / ml. After inoculating the algae seed, add disinfectant to ensure that the effective chlorine concentration in the algae reaches 5mg / L, and let the water stand for 1 hour. Pour the algae solution: Pour the upper algae solution into another disinfected and sterilized glass container, and leave the protozoa and sediment in the original container and discard; Neutralization of residual chlorine: Sodium thiosulfate was added to the transferred algae solution at a concentration of 0.5 mg / L to neutralize the residual available chlorine. Secondary disinfection: 48 hours after the initial treatment, add sodium hypochlorite or bleaching powder or bleaching powder essence containing available chlorine for disinfection again. After the water body is allowed to stand, observe whether there is precipitation. If there is, it is necessary to replace the container again. If not, there is no need to replace it. Then add the corresponding concentration of sodium thiosulfate; After the second treatment, the algae are regularly checked daily using a microscope for the presence of protozoa. If protozoa are still present, the algae are disinfected again to confirm their condition and purity. Subsequently, culture medium is added to the algae solution to provide the necessary nutrients and the algae culture is continued. After 5 to 6 days, when the color of the Chlorella in the culture medium changes from light green and slightly yellow to emerald green to dark green, it meets the needs of expansion and enters the harvesting stage. Use a filter to remove the Chlorella and clean and process it.
2. The method for removing protozoa from a Chlorella seed bottle according to claim 1, characterized in that: The disinfectant is any one or two of sodium hypochlorite, bleaching powder or bleaching powder essence.
3. The method for removing protozoa from a Chlorella seed bottle according to claim 1, characterized in that: The ratio of the nutrient solution is 74.8 mg / L of sodium nitrate, 22.5 mg / L of urea, 4.4 mg of sodium dihydrogen phosphate, and 3.9 mg / L of ferric citrate.